Inhibition of polyisoprenoid and glycoprotein biosynthesis causes abnormal embryonic development.

Compactin, a potent inhibitor of polyisoprenoid biosynthesis, induces abnormal gastrulation during sea urchin development at concentrations that have no effect on earlier embryonic development or on macromolecular synthesis. Three lines of evidence suggest that the developmental lesion caused by compactin results from inhibition of dolichol biosynthesis and a concomitant inhibition in the biosynthesis of the oligosaccharide chains of N-linked glycoproteins. (i) Embryos cultured in the presence of compactin gastrulate normally when supplemented with dolichol alone, whereas supplementation with cholesterol or coenzyme Q or both does not prevent the compactin-induced developmental lesion. (ii) Exogenously supplemented [3H]dolichol is incorporated into a compound with the chromatographic properties of oligosaccharide-pyrophosphoryldolichol. (iii) Embryos cultured in the presence of compactin exhibit a decreased capacity to synthesize mannose-labeled glycolipids and N-linked glycoproteins. This decrease in synthesis is abolished if the embryos are cultured in the presence of dolichol along with compactin.


Relationship between movement and aggregation of centrioles in syncytia and formation of microtubule bundles.

Previous reports from this laboratory have provided evidence suggesting that microtubules and 10-nm filaments serve both cytoskeletal and force-generating functions in the intracellular movement and positioning of nuclei in syncytia. It has been found that, during the process of cell fusion and nuclear migration in syncytia induced by the paramyxovirus simian virus 5, centrioles are transported in the cytoplasm and form large aggregates. These aggregates are usually found in regions adjacent to rows of aligned nuclei and large bundles of microtubules and 10-nm filaments. Colchicine prevents the translocation and aggregation of centrioles, but cytochalasin B has little effect on this process. These results suggest that the same cytoskeletal elements that are involved in nuclear migration and positioning--i.e., microtubules and 10-nm filaments--are also involved in the transport of centrioles. The possibility that aggregates of centrioles may serve as centers for the organization of microtubules and 10-nm filaments into the large bundles observed in the syncytia is discussed.


In vitro synthesis of a putative precursor of mitochondrial ornithine transcarbamoylase.

Ornithine transcarbamoylase (OTCase; ornithine carbamoyltransferase; carbamoyl phosphate:L-ornithine carbamoyltransferase, EC 2.1.3.3), a major mitochondrial matrix enzyme in ureotelic animals, is synthesized on cytoplasmic ribosomes and translocated across both mitochondrial membranes to the matrix. In an attempt to identify the primary translation product (or an early intermediate) that is the substrate for this transport process, we translated rat liver polysomal RNA in vitro by using the rabbit reticulocyte lysate system. Immunoprecipitation of the [35S]methionine-labeled translation mixture was performed by using monospecific OTCase antiserum and the immunoadsorbent Staphylococcus aureus. Approximately 0.3% of total trichloroacetic acid-insoluble 35S-labeled material was specifically precipitated. Analysis of the precipitate by fluorography of a dried sodium dodecyl sulfate/polyacrylamide gel showed a single major translation product whose mobility corresponded to a polypeptide of 43,000 daltons, a value approximately 4000 daltons greater than that noted for the "mature" OTCase subunit isolated from rat liver. This translation product was not precipitated by preimmune rabbit serum, and excess unlabeled mature OTCase competed with it for interaction with OTCase antiserum. These results suggested that rat liver OTCase, like a number of other cytoplasmically synthesized organellar proteins, is initially made as a larger precursor that contains an amino acid sequence necessary to confer on OTCase its transport properties. The potential application of these findings to the study of inherited complete OTCase deficiency in humans is discussed.


Phosphoethanolamine as a growth factor of a mammary carcinoma cell line of rat.

We have identified phosphoethanolamine as the pituitary-derived growth-promoting material that specifically stimulates the rat mammary carcinoma cell line 64-24. We have been studying the growth characteristics of the 64-24 cell line, which was isolated from a highly hormone-dependent tumor and which retains in culture many characteristics of the original tumor. Previously, crude bovine pituitary extract was shown to contain a significant amount of growth-stimulating activity for these cells, and a growth factor from this extract was purified to homogeneity. This report describes the identification of the growth factor as phosphoethanolamine. Further, the biological activity of phosphoethanolamine was found to be virtually identical to that of the purified growth factor. A possible role of phosphoethanolamine in the growth of mammary tumor cells as well as of normal mammary epithelial cells and other tissues is discussed.


Purification of cytoplasmic tubulin and microtubule organizing center proteins functioning in microtubule initiation from the alga Polytomella.

Cytoplasmic tubulin and the microtubule organizing centers (MTOCs) for the cytoskeletal microtubule system of the flagellate Polytomella have been isolated. The isolated MTOCs serve as sites for the in vitro assembly of the purified tubulin protein. The major proteins (four polypeptides of molecular weights 190,000-210,000) functioning in this assembly have been extracted from the MTOCs and purified. Kinetic studies and experiments with in vivo 35S-labeled MTOC proteins (or 35S-labeled tubulin) demonstrate that these proteins function specifically in microtubule initiation and do not contribute to microtubule elongation. The results indicate that microtubule assembly in vivo is controlled by microtubule initiating proteins associated with the organelles termed MTOCs.


Early termination of heterogeneous nuclear RNA transcripts in mammalian cells: accentuation by 5,6-dichloro 1-beta-D-ribofuranosylbenzimidazole.

Labeling of RNA in isolated HeLa cell nuclei in vitro reveals an abundance of short RNA chains made by RNA polymerase II. These short chains were initiated prior to isolation of the nuclei. The short abundant chains are increased in amount in nuclei isolated from cells treated with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB). Kinetic evidence indicates that the bulk of the putative heterogeneous nuclear RNA (hnRNA) precursor molecules that are terminated early in vivo are terminated approximately 100-300 nucleotides from sites of initiation. DRB increases the frequency of early termination, but there is a fraction of hnRNA precursor molecules whose elongation is not affected by DRB. Heparin is useful in studies of hnRNA transcription in isolated nuclei because it enhances chain elongation.


Oncogenic transformation of mammalian cells in vitro with split doses of x-rays.

An established line of mouse fibroblasts, C3H/10T1/2 cells, was used for the assessment in vitro of oncogenic transformations caused by single and split doses of x-rays. The shape of the dose-response relationship was determined over the range from 0.1 to 10 Gy. It was found that splitting the x-ray dose into two equal fractions, separated by 5 hr, led to a reduction in transformation frequency at doses above 1.5-2 Gy but to an enhancement of transformation at lower doses. The observations reported cast doubt on the assessment of human cancer risk at lo dose levels by a linear extrapolation from available high-dose data from the Japanese atomic bomb survivors or from persons exposed for medical purposes.


Effects of opiates and demographic factors on DNA repair synthesis in human leukocytes.

DNA repair synthesis in leukocytes stressed by far UV irradiation was studied in 90 normal individuals, 38 street-heroin addicts, and 18 methadone maintenance patients. Age, sex, coffee use, and alcohol use had no significant effect on the maximal repair synthesis response of the control subjects, but smoking tobacco significantly decreased the mean response and variance when compared with nonsmoking controls. Heroin addiction had an even more pronounced negative effect, and this may be related to the high rate of chromosome aberrations found in this population. Half of the addicts tested were incapable of repairing UV fluences one-quarter as large as those repaired by the control subjects (5 J/m2 and 20 J/m2, respectively) in the 2-hr assay period. Long-term methadone treatment ameliorated the effects of the street heroin, just as it resulted in a decrease of the chromosome aberration frequency.


Regional assignment of the steroid sulfatase-X-linked ichthyosis locus: implications for a noninactivated region on the short arm of human X chromosome.

The expression of steroid sulfatase (SS; sterol-sulfatase; sterol-sulfate sulfohydrolase, EC 3.1.6.2), a microsomal enzyme that catalyzes the hydrolysis of a variety of 3beta-hydroxysteroid sulfates, was evaluated in mouse-human hybrid clones. The mouse parental line, A9, was found to have little SS as determined by activity measurements. Human SS can be separated from mouse SS by electrophoresis. Two human fibroblast lines, one carrying an X/13 translocation [46,X,t(X;13)(p22;q12)] and the other carrying an X/20 translocation [46,X,t(X;20)(Xp20q;Xq20p)] were used as the human parental lines. Several independently derived hybrid clones from each of the two fusion experiments were analyzed for the expression of human SS by activity measurements and electrophoresis. Cytogenetic analyses were done on these hybrid clones at the same passage level. The results showed that the expression of human SS in these cell hybrids was concordant only with the presence of the distal half (p22-->pter) of the short arm of the human X chromosome, thus assigning the locus for SS to Xp22-->Xpter. Earlier studies have shown that the deficiency of SS is the basis for the dermatologic condition X-linked ichthyosis, the gene for which is known to be located approximately 10 centimorgans from the Xg blood group locus. The localization of SS on the X chromosome indicates that Xg locus may be on the short arm of X and possibly on its distal half. The Xg locus is thought to escape X-inactivation in man, and recent investigations suggest that the SS locus also escapes X-inactivation. Our results thus provide evidence for the location of an apparently noninactivated site on the distal half of the short arm of the human X-chromosome that contains the locus for SS and possibly the Xg locus.


Antibody-induced modulation of Friend virus cell surface antigens decreases virus production by persistent erythroleukemia cells: influence of the Rfv-3 gene.

The Rfv-3 gene was found to influence the level of Friend leukemia virus production in spleens of leukemic mice later than 30 days after virus inoculation. Rfv-3r/s mice [(B10.A X A)F1 and (B10.A X A.BY)F1] had decreased spleen virus levels 30-90 days after virus inoculation compared to Rfv-3s/s mice [A.BY, A, BALB.B, and (BALB/c X A)F1)]. In (B10.A X A)F1 X A backcross mice the spleen virus titer segregated with the level of viremia. The Rfv-3 gene appeared to act by controlling anti-Friend virus antibody production. The interaction of antiviral antibody with infected cells led to a decrease in release of infectious virus by late leukemic spleen cells in Rfv-3r/s mice to 1/300th that in Rfv-3s/s mice. This decrease in virus release appeared to be due to interference with the virus budding process due to antibody-mediated modulation of virus-induced cell surface antigens.


Assignment of human beta-, gamma-, and delta-globin genes to the short arm of chromosome 11 by chromosome sorting and DNA restriction enzyme analysis.

Normal human metaphase chromosomes isolated from fibroblasts were resolved into 14 peaks based on total Hoechst 33258 fluorescence and sorted with the fluorescence-activated cell sorter. The chromosomal DNA was extracted and characterized by EcoRI analysis. As expected, analysis of the peak containing chromosomes 16 and 18 detected the alpha-globin genes and of the peak containing chromosomes 9, 10, 11, and 12 detected the beta-, gamma-, and delta-globin genes. Translocations were then used to localize further the beta-, gamma-, and delta-globin genes. The first translocation t(11;22)(q25;q11), which moved nearly all of chromosome 11 to a different peak, confirmed that the beta-, gamma-, and delta-globin genes are on this chromosome. The second, t(4;11)(q25;q13), which moved the distal portion of the long arm of chromosome 11 to a new peak, showed that the genes are not in this segment. The third, t(X;11)(q11;p13), moved the distal region of the short arm of chromosome 11 to a peak which now contained the beta-, gamma-, and delta-globin genes. Therefore, the beta-, gamma-, and delta-globin genes residue on the distal portion of the chromosome 11 short arm including bands p13, p14, and p15. This sorting method may be used generally to assign other genes to chromosomal segments of the entire chromosome complement.


Fv-2 locus controls expression of Friend spleen focus-forming virus-specific sequences in normal and infected mice.

We have recently demonstrated that normal hemopoietic cells express RNA sequences that are homologous to sequences specific for the Friend erythroleukemia virus genome [Bernstein, A., Gamble, C., Penrose, D. & Mak, T. W. (1979) Proc. Natl. Acad. Sci. USA 76, 4455-4459]. In this communication, we report that the Fv-2 locus, the major genetic determinant controlling host susceptibility to erythroleukemia induction by Friend leukemia virus, also controls the expression of endogenous sequences related to the replication-defective component of Friend leukemia virus, Friend spleen focus-forming virus (SFFV), in normal uninfected mice. Two independent congeneic pairs of mice [C57BL/6 (B6) and B6.S; B6 and B6.C(H-7(b))], differing only in a small region of the mouse genome including the Fv-2 locus, were used for this purpose. In both cases, molecular hybridization analysis indicated that the presence of SFFV-related RNA sequences in normal mice was associated with the Fv-2(s) allele: bone marrow or spleen cellular RNA from Fv-2(rr) B6 mice contained no detectable SFFV-related sequences, whereas their congeneic Fv-2(ss) pairs contained relatively high levels of these RNA sequences. The absence of these RNA sequences in Fv-2(rr) mice was not due to deletion of these sequences from the DNA of Fv-2(rr) mice. Repopulation of lethally irradiated Fv-2(rr) mice with syngeneic Fv-2(rr) bone marrow cells did not lead to any increase in the levels of these SFFV-related RNA sequences, suggesting that the expression of these sequences is still reduced or inhibited in actively cycling Fv-2(rr) hemopoietic cells. Infection with Friend leukemia virus resulted in the appearance of high levels of RNA homologous to SFFV-specific sequences in the leukemic spleens of B6.S (Fv-2(ss)) mice, whereas these cellular RNA sequences could not be detected in the spleens of Friend virus-infected B6 (Fv-2(rr)) mice. The demonstration that the same gene locus controls both the expression of exogenous SFFV-specific sequences and erythroleukemia induction by Friend leukemia virus suggests that these sequences may be necessary for erythroleukemic transformation. In addition, the finding that the Fv-2 gene locus controls the expression of endogenous SFFV-related sequences suggests that these sequences may also be involved in normal hemopoiesis.


Specific-locus test shows ethylnitrosourea to be the most potent mutagen in the mouse.

Use of the specific-locus test to measure the frequency of transmitted gene mutations induced in mouse spermatogonia has shown ethylnitrosourea to be by far the most potent mutagen yet discovered in the mouse. The dose used, 250 mg/kg, gave a mutation rate 5 times as high as had been obtained with 600 R, the most effective acute dose of x-rays. Compared to procarbazine, heretofore the most mutagenic chemical known in the mouse, ethylnitrosourea proved to be 15 times more mutagenic than the peak effect obtained with the most effective dose of procarbazine. Because of its high mutagenicity, ethylnitrosourea can serve as a model compound in exploring the effect of such factors as dose response, dose fractionation, sex, and cell stage on the mutagenic action of a chemical. Ethylnitrosourea is clearly the mutagen of choice for the production of any kind of desired new gene mutations in the mouse.


DNA-mediated gene transfer of a circular plasmid into murine cells.

We have used DNA-mediated gene transfer to introduce a recombinant plasmid containing the human beta-globin gene (H beta 1) into cells of a mouse tissue culture line, Ltk-. DNA isolated from independent transfer lines was analyzed by restriction endonuclease digestion, gel electrophoresis, modified Southern blotting, and filter hybridization using H beta 1 as a probe. H beta 1 sequences were present in 80% of the lines at 1-30 copies per cell. Many of the lines gave a hybridization pattern indicative of H beta 1 sequences integrated into high molecular weight DNA. DNA from three cell lines, digested with several restriction enzymes, produced a pattern providing evidence for the presence of circular H beta 1 molecules in the murine recipient cells.


Regulation of the amplification C3 convertase of human complement by an inhibitory protein isolated from human erythrocyte membrane.

An activity that is inhibitory to the properdin-stabilized amplification C3 convertase (C3b,Bb,P) was solubilized from human erythrocyte (E(hu)) membranes by Nonidet P-40 and purified to homogeneity. The inhibitory membrane glycoprotein had an apparent M(r) of 1-1.2x10(6) on gel filtration in the presence of Nonidet P-40. On sodium dodecyl sulfate/polyacrylamide gel electrophoresis it presented a single stained band with an apparent M(r) of 205,000, with or without prior reduction of disulfides. The inhibitory protein of the E(hu) membrane produced a dose-related, first-order decay of C3b,Bb,P function on sheep erythrocytes (E(s)) and released (125)I-labeled Bb from these sites, indicating a mechanism of inhibition by decay-dissociation of the amplification C3 convertase. The 50% inhibitory dose of the E(hu) membrane protein was not altered by removal of sialic acid from the E(s) bearing C3b,Bb,P sites. E(hu) membrane protein also serves as a cofactor for C3b inactivator-induced cleavage of the alpha polypeptide chain of C3b. Thus, the inhibitory membrane protein can abrogate the activity of amplification convertase sites that have formed and also can prevent generation of such sites by augmenting irreversible inactivation of C3b.Discrimination between cells by the alternative complement pathway occurs after initial deposition of C3b and is related to the modulation by surface constituents of the capacity of bound C3b to function as a subunit of the amplification C3 convertase. The existence in the E(hu) membrane of a protein that can impair the functions of membrane-bound C3b and C3b,Bb,P could represent a molecular basis for preventing inappropriate self-recognition.


Evidence for a two-domain structure of the terminal membrane C5b-9 complex of human complement.

Lipid vesicles carrying the purified membrane C5b-9 complex [C5b-9(m)] of complement were analyzed immunochemically and in the electron microscope after treatment with a combination of trypsin and alpha-chymotrypsin. Under reducing conditions, the externally oriented annulus was removed. The remaining part of the C5b-9(m), representing approximately half of the total mass of the macromolecular complex, was visualized in the electron microscope as a hollow cylindrical structure with walls of 1-nm thickness. This structure remained tenaciously attached to the lipid bilayer, projecting 8-9 nm from the external membrane surface into the aqueous environment. Cleavage of C5b-9(m) by proteolysis and reduction resulted in a sharp reduction of tis antigenic determinants. One hydrophilic protease-resistant C5 derivative was released from the membrane and recovered in the fluid phase. The membrane-bound residue almost totally lacked antigens precipitable with antisera to C5, C6, C9, and C5b-9(m).


Primary defect of insulin receptors in skin fibroblasts cultured from an infant with leprechaunism and insulin resistance.

Insulin binding to insulin receptors, on skin fibroblasts established in culture from an infant with insulin resistance and clinical features of leprechaunism was markedly decreased in comparison with cultures from an age-matched control. By contrast, the binding of epidermal growth factor, a polypeptide growth factor chemically unrelated to insulin, to patient's and control fibroblasts was indistinguishable. The selective defect in insulin binding to patient's fibroblasts was reflected in an impaired ability of insulin to stimulate 2-deoxyglucose uptake. These results most likely indicate a primary genetic defect of insulin receptors.


Enhancement of hexose uptake in human polymorphonuclear leukocytes by activated complement component C5a.

The polymorphonuclear leukocyte (PMNL) depends on glucose as a source of energy for motility, chemotaxis, phagocytosis, and bactericidal activity. Activated complement (C5a) at low concentrations stimulates carrier-mediated carbohydrate transport in PMNLs as measured by the uptake of 2-deoxy-D-[(3)H]glucose. Human PMNLs were preincubated at 37 degrees C for 15 min with zymosan-activated human serum or various purified preparations of human C5a. A concentration-dependent increase in deoxyglucose transport (>700% of control) into PMNLs occurred with all test substances. Reaction was linear for 30 min, and uptake of deoxyglucose followed saturation kinetics. C5a caused a decrease in the K(m) for deoxyglucose, from 0.53 to 0.11 mM, without altering the V(max) (44 nmol/30 min per 5 x 10(6) PMNLs in control and 46.6 with C5a). The optimal concentration of C5a for enhanced carrier-mediated transport of deoxyglucose was similar to that which promoted optimal chemotaxis. Activated serum from C5-deficient mice had little or no effect on deoxyglucose transport whereas that from normal syngeneic mice enhanced deoxyglucose transport. C5a did not enhance deoxyglucose transport into isolated erythrocytes, platelets, or lymphocytes. The deoxyglucose within the cell was primarily in the phosphorylated form, and hexokinase activity was not increased in PMNLs stimulated with C5a, indicating that hexokinase was not rate limiting and that enhanced transport was the mechanism of the C5a activity. Insulin at physiologic concentration (10 ng/ml) had no effect on deoxyglucose transport in PMNL and did not act as a competitive inhibitor of C5a. This insulin-like bioactivity could be detected with the amount of C5a that would be present after activation of 0.1-0.5% of the C5 in 1 ml of serum. This suggests that uptake of [(3)H]deoxyglucose by PMNLs might serve as a highly sensitive test for activation of the fifth component of complement.


Spontaneous tumors in Sprague-Dawley and Long-Evans rats and in their F1 hybrids: carcinogenic effect of total-body x-irradiation.

Rats frequently develop various tumors, many of them malignant; the majority of tumors in the females develop in the mammary glands. In primary spontaneous tumors and lymphomas virus particles cannot be found on electron microscopic examination; transmission of the tumors by filtered extracts has not been successful. In our colonies of Sprague-Dawley rats the incidence of tumors was 22% in females and 5% in males; in Long-Evans rats the incidence of tumors and 28% in females and 10% in males. In (Sprague-Dawley x Long-Evans) F1 hybrids the incidence of tumors was 67% in females and 32% in males, about twice as high as in the parental strains. Fractionated total-body x-irradiation (150 rads five times at weekly intervals) (1 rad = 0.01 gray) increased the incidence of tumors in Sprague-Dawley rats from 22% to 93% in females and from 5% to 59% in males. In Long-Evans rats, irradiation increased the incidence of tumors from 28% to 63% in females and from 10% to 42% in males. The incidence of malignant tumors was almost twice as high in irradiated Sprague-Dawley and Long-Evans rats as compared with nonirradiated animals of the same strains. Partial shielding during irradiation had no significant effect on the incidence or on the forms of tumors developing in the irradiated animals. In striking contrast to results of experiments carried out on mice, the incidence of leukemia and lymphomas was not increased in the irradiated rats, as compared with control animals.


Pyrolysis products from amino acids and protein: highest mutagenicity requires cytochrome P1-450.

Pyrolysis products of proteins and amino acids are highly mutagenic, but metabolism of these chemicals by rat liver subcellular fractions is known to be required for production of the mutagenic intermediates. We examined the mutagenesis of seven purified pyrolysis products from tryptophan, lysine, glutamic acid, and soybean globulin with Salmonella typhimurium strain TA98 in the presence of liver fractions from genetically "responsive" C57BL/6N and Ah(b)/Ah(d) or "nonresponsive" DBA/2N and Ah(d)/Ah(d) mice that had been pretreated in vivo with benzo[a]pyrene. For all pyrolysis products tested, mutagenesis is 2-fold to more than 1000-fold greater with C57BL/6N and Ah(b)/Ah(d) than with DBA/2N or Ah(d)/Ah(d) liver fractions. A sucrose density gradient assay for detecting the Ah regulatory gene product, the receptor, was studied with C57BL/6N hepatic cytosol. At levels 100 times in excess of [1,6-(3)H]2,3,7,8-tetrachlorodibenzo-p-dioxin, nonlabeled 2,3,7,8-tetrachlorodibenzo-p-dioxin, 3-methylcholanthrene, and beta-naphthoflavone (inducers of cytochrome P(1)-450) are able to displace the radioligand from its hepatic cytosolic receptor; four pyrolysates from tryptophan, glutamic acid, and soybean globulin did not have this capacity. These data indicate that the pyrolysis products tested, although not effective as inducers of cytochrome P(1)-450, are most mutagenic when metabolized by P(1)-450. Potent P(1)-450 inducers-present in pyrolysates during the combustion process-might be present in quantities insufficient to initiate mutagenesis or carcinogenesis but might have a synergistic action, or act as "comutagens" or "cocarcinogens," with the N-containing heterocyclic pyrolysis products. A quantitative relationship between mutagenic and carcinogenic potency of these pyrolysis products remains, however, to be demonstrated.


Galactosamine-induced sensitization to the lethal effects of endotoxin.

Treatment of rabbits, rats, and mice with D-galactosamine increased their sensitivity to the lethal effects of lipopolysaccharide several thousand fold. The susceptibility of the animals was highest when the lipopolysaccharide was injected together with galactosamine and decreased successively when injection was carried out 1, 2, and 3 hr later. Sensitization was absent when the lipopolysaccharide was administered 1 hr before or 4 hr after galactosamine. The onset of lethality after treatment with galactosamine and lipopolysaccharide occurred faster than with lipopolysaccharide alone; usually all animals died 5-9 hr later. The galactosamine-induced sensitization to lipopolysaccharide could be reversed by uridine which is known to inhibit the early biochemical alterations induced by the amino sugar in the hepatocytes. Although galactosamine is known to exhibit hepatotoxic activity inducing ultimate necrosis of the hepatocytes, the data so far suggests that the sensitization to lipopolysaccharide is related only to the early metabolic effects of the hexosamine.


Persistence of circadian rhythmicity in a mammalian hypothalamic "island" containing the suprachiasmatic nucleus.

The experimental work described tested the prosposition that the suprachiasmatic nucleus of the hypothalamus is an autonomous circadian pacemaker. Simultaneous recording from two extracellular electrodes indicated neural (multiple unit) activity at two sites in the brain, one of which is in or near the suprachiasmatic nucleus and the other in one of many other brain locations. Both sites in intact rats displayed clear circadian rhythmicity of spontaneous neural activity. In experimental animals, a Halasz knife was used to create an island of hypothalamic tissue that contained the suprachiasmatic nuclei. In such animals that were also blinded by bilateral ocular enucleation, circadian rhythmicity was lost at all brain locations recorded outside the island, but it persisted within the island that contained the suprachiasmatic nuclei. The rhythmicity of the island is thus not dependent on afferent inputs from elsewhere in the brain.


Blockage of narcotic-induced dopamine receptor supersensitivity by cyclo(Leu-Gly).

We have previously reported that the administration of cyclo(Leu-Gly) to mice prior to morphinization blocked the development of tolerance to the analgesic effects of morphine as well as the development of some signs of physical dependence. In the present series of experiments, the effect of the same peptide treatment on changes in dopamine receptor sensitivity induced by chronic morphine treatment were determined. Changes in dopamine receptor sensitivity were determined by measuring (i) the effect of the dopamine agonist apomorphine on locomotor activity and (ii) the hypothermic response to another dopamine agonist, piribedil. Mice that had received the chronic morphine treatment were found to require significantly less apomorphine to produce an increase in locomotor activity, and they exhibited a significantly greater hypothermic response to piribedil than did morphine-naive mice. The injection of 0.2 mumol of cyclo(Leu-Gly) per mouse 2 hr prio to morphine treatment prevented this increased response to both dopamine agonists. Administration of the peptide after the tolerance and dependence had developed did not alter morphine tolerant and dependent states states or the enhanced response to apomorphine or piribedil. It is concluded that dopamine receptor supersensitivity may be involved in the development of narcotic tolerance and physical dependence.


Interrelationships between ganglionic acetylcholinesterase and nonspecific cholinesterase of the cat and rat.

When homogenates of cat or rat superior cervical ganglia in Krebs-Ringer solution were incubated at 37 degrees C, the ensuing decrease in acetylcholinesterase (acetylcholine acylhydrolase, EC 3.1.1.7) activity was increased significantly by prior administration in vivo of tetramonoisopropylpyrophosphotetramide at doses that produced selective alkylphosphorylation of butyrylcholinesterase or propionylcholinesterase. These findings are consistent with the proposal that the latter enzymes are posttranscriptional precursors of acetylcholinesterase. Results of similar studies with homogenates of ganglia in water or in M NaCl/1% Triton X-100 were inconclusive, as were those of heat-inactivation studies and immunoprecipitation of the enzymes.


Treatment of nerve impulse data for comparison with theory.

A procedure is given for the comparison of nerve impulse data with model predictions. This method utilizes information in the nerve impulse train that is ignored by the post-stimulus-onset histogram and thereby gives an improved signal-to-noise ratio. Comparison of observed responses in the Limulus retina with predictions derived from a detailed model gives good agreement.


Carbon monoxide binding to iron porphyrins.

The carbon monoxide affinities of iron complexes of meso-tetra (alpha, alpha, alpha, alpha-o-pivalamidophenyl)porphyrin (the "picket fence" porphyrin) and of a "picket fence" porphyrin derivative with an appended axial base have been measured in solution and compared with the CO affinities of various hemoproteins. The model complexes bind CO with much greater affinity than normal hemoproteins; the role of the steric bulk of distal residues in lowering the CO affinities of the hemoproteins is discussed. The significance of this lowered CO affinity is described with regard to endogenous CO. A discussion of mutant hemoglobins lacking distal residues that sterically inhibit the binding of CO is presented. The use of pressure units versus concentration units in equilibrium expressions is analyzed.


Structure of carboxymyoglobin in crystals and in solution.

The configuration of the heme-carbonyl group upon binding of carbon monoxide to sperm whale myoglobin (Mb) in crystals is evaluated on the basis of infrared spectroscopic methods. Multiplets of the totally symmetric C-O stretching mode are observed for the heme-bound ligand near 1933, 1944, and 1967 cm-1, corresponding to three different heme-carbonyl conformers. Variations in the relative proportions of these conformers can be induced by incorporation of small fractions of metMb or deoxyMb into MbCO crystals. The configuration of the iron-carbonyl with respect to the immediate coordination environment of the heme iron is assigned for each v(CO) stretching frequency on the basis of a detailed comparison of the three-dimensional structures of the heme environments of MbCO, metMb, and deoxyMb defined by crystallographic methods. The structures of the three heme-carbonyl conformers account for the v(CO) infrared absorption bands that can be observed for MbCO in solution.


A possible folding pathway of bovine pancreatic RNase.

A theoretical pathway for the folding of RNase into its native conformation is derived from the contact map computed from crystallographic coordinates. The pathway is based on the hypothesis of Tanaka and Scheraga, according to which localized conformations stabilized by short- and medium-range interactions form before those conformational features that are stabilized primarily by long-range interactions. The pathway deduced from the contact map agrees with experimental information on intermediates detected in the thermal unfolding of RNase and in immunochemical studies on the formation of stable antigenic sites when deduced RNase is oxidized with glutathione. Ambiguities in the interpretation of the contact map are resolved by the combination of structural information contained in the contact map and experimental information.


Phosphorylation of specific, distinct proteins in synaptosomes and axons from squid nervous system.

Synaptosomes and axons from squid were incubated with [gamma-(32)P]ATP or [(32)P]orthophosphate and specific, distinct proteins were found to be labeled in each preparation. In axoplasm, only the major 200,000 M(r) neurofilament protein and a specific protein of approximately 400,000 M(r) were labeled, as reported previously [Pant, H. C., Shecket, G., Gainer, H. & Lasek, R. J. (1978) J. Cell Biol. 78, R23-R27]. These results were independent of whether the cosubstrates were (32)PO(4) (2-) or [gamma-(32)P]ATP. However, synaptosomes lacked the 200,000 M(r) neurofilament protein and several lower molecular weight proteins were labeled instead, the most prominent being a 47,000 M(r) species. [gamma-(32)P]ATP was much more effective in labeling the 47,000 M(r) species than (32)PO(4) (2-). Synaptosomes also contained a distinct 250,000 M(r) protein species which, however, was not labeled. The protein kinase activity in synaptosomes was sensitive to various pharmacological agents, depending on whether the labeled phosphate came directly from ATP or orthophosphate. Carbonyl cyanide p-trifluoromethoxyphenyl hydrazone, a mitochondrial H(+) uncoupler, almost completely inhibited incorporation of (32)P into protein with (32)PO(4) (2-) as cosubstrate, as expected, but produced only 32% inhibition with [gamma-(32)P]ATP as cosubstrate. The activity could be augmented by incubating synaptosomes in a calcium-free medium and could be suppressed by increasing intrasynaptosomal Ca(2+) with A23187, a Ca(2+) ionophore. The latter effect was more prominent with (32)PO(4) (2-) than with [gamma-(32)P]ATP as cosubstrate. Depolarizing agents such as veratridine and high K(+) also suppressed activity, and the veratridine effect was completely reversed by tetrodotoxin or by omission of Ca(2+) when [gamma-(32)P]ATP was used, and partially reversed when (32)PO(4) (2-) was used. We conclude that the morphological transformation of an axon into a terminal is accompanied by significant changes in protein and phospho-protein composition that may be related to synaptic transmission.


Electron microscopic visualization of N-acetoxy-N-2-acetylaminofluorene binding sites in ColE1 DNA by means of specific antibodies.

ColE1 DNA has been allowed to react in vitro with N-acetoxy-N-2-[14C]acetylaminofluorene in the range of 0-15 N-2-[14C]acetylaminofluorene residues bound per molecule of DNA, at the C8 of guanine residues. Purified rabbit antibodies to both N-2-(guanosine-8-yl)-acetylaminofluorene and native DNA that had reacted with N-acetoxy-N-2-acetylaminofluorene were shown by electron microscopy to recognize specifically the acetylaminofluorene-modified ColE1 DNA. The antibodies bound to DNA were visualized either per se or after reaction with goat anti-rabbit immunoglobulins coupled with ferritin. There was a linear relationship between the average number of antibodies bound per DNA molecule and the number of N-2-(deoxyguanosine-8yl)-acetylaminofluorene residues per DNA molecule. The slope of this straight line was equal to 0.4. Due to the bivalence of the immunoglobulins one would expect a value of 0.5; we actually observed an important fraction of the bound antibodies crosslinking two parts of the same (or of another) DNA molecule.


Diadenosine 5',5'''-P1,P4-tetraphosphate, a ligand of the 57-kilodalton subunit of DNA polymerase alpha.

By equilibrium dialysis a disadenosine 5',5'''-P1,P2-tetraphosphate (Ap4A) binding activity is shown to be present in mammalian cells. The Ap4A binding activity copurifies with DNA polymerase alpha during the isolation procedure, which includes chromatography on phospho-, DEAE-, and DNA-cellulose; gel filtration; sucrose gradient centrifugation; and electrophoresis in nondenaturing polyacrylamide gels. After these purification steps, DNA polymerase alpha appears to be homogeneous in nondenaturing polyacrylamide gels. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of such a purified DNA polymerase alpha preparation reveals seven distinct protein bands with apparent Mrs of 64,000, 63,000, 62,000, 60,000, 57,000, 55,000, and 52,000. By affinity labeling, the protein with Mr 57,000 has been shown to be the Ap4A-binding constituent of DNA polymerase alpha. The binding activity of DNA polymerase alpha for Ap4A is highly specific because neither structural analogs nor several other adenine nucleotides compete effectively with Ap4A for its binding site. The Ap4A binding site is lost in neuronal cells during maturation of rat brains concomitantly with the loss of DNA polymerase alpha and mitotic activity in those cells. From these results, DNA polymerase seems to be the intracellular target of Ap4A. This is discussed in respect to the recently reported of Ap4A to trigger DNA replication in quiescent mammalian cells [Grummt, F. (1978) Proc. Natl. Acad. Sci. USA 75, 371-375].


Trypsin liberates an arginine vasopressin-like peptide and neurophysin from a Mr 20,000 putative common precursor.

Although the hypothesis that vasopressin and its associated neurophysin are synthesized together in one macromolecular common precursor was put forward more than a decade ago, direct conformation of this hypothesis has been lacking. A [35S]cysteine-labeled putative precursor for vasopressin-related neurophysin (Mr 20,000, pI 6.1) has been isolated from the supraoptic nuclei of rats. This precursor was subjected to limited proteolysis with trypsin which produced a Mr 10,000 protein and peptide products. The former was identified as neurophysin on the basis of its pH-dependent affinity for vasopressin and its behavior in isoelectric focusing systems (pI 4.6-4.8). The tryptic peptides proved to be vasopressin-like because they: (i) were rich in cysteine, (ii) comigrated with vasopressin on gel filtration columns in 6 M guanidine HCl, (iii) bound to a neurophysin-Sepharose affinity column at pH 5.7, and (iv) were recognized by antibodies against vasopressin. These data on the Mr 20,000, pI 6.1 protein represent direct experimental evidence for a candidate for the common precursor of vasopressin and neurophysin. We propose that this common precursor be called "propressophysin."


Binding of cis- and trans-dichlorodiammineplatinum(II) to the nucleosome core.

The binding of the antitumor drug cis-dichlorodiammineplatinum(II) and its inactive trans isomer with the nucleosome core particle has been investigated. Kinetic studies show that platinum binding increases with incubation time, from a few bound platinum atoms per nucleosome core in the first 0.5 hr to 40-50 after 40 hr. There is no crosslinking or dissociation of nucleosome cores upon platinum binding, as revealed by sedimentation velocity measurements. Electrophoresis through 0.1% sodium dodecyl sulfate/18% polyacrylamide gels after platinum binding reveals striking differences in the DNA and protein band patterns for the two isomers. cis-Dichlorodiammineplatinum(II) binds first to the DNA, retarding and spreading its migration in the gel. A comparison study with the 146-base-pair nucleosome core DNA showed the binding to be little affected by the presence of the histone octamer. The trans complex, on the other hand, produces DNA-histone and histone-histone crosslinks that only appear for the cis isomer after long incubation times. The protein-protein crosslinks were reversed by soaking the gel in cyanide solution to form [Pt(CN)(4)](-2). Subsequent two-dimensional gel electrophoresis revealed that trans-dichlorodiammineplatinum(II) forms specific crosslinks between histone protein pairs H3 and H2a and H2b and H4 in the nucleosome core. The occurrence of DNA-protein crosslinks was demonstrated by treating the platinum/nucleosome core reaction mixtures with a protease or with nucleases prior to electrophoresis and observing changes in the gel patterns. Platinum was located in the gels through autoradiography using (195m)Pt-labeled complexes. This work clearly demonstrates the greater propensity of trans-dichlorodiammineplatinum(II) to form histone-histone and histone-DNA crosslinks compared with the antitumor active cis isomer, which binds first to the DNA and only forms crosslinks to the histones when the nucleosome core is heavily loaded with platinum.


Internal organization of long repetitive DNA sequences in sea urchin genomes.

In keeping with earlier reports, we have found that reassociated long repeat DNA from sea urchins is thermostable, indicating the absence of evolutionarily diverged families of repeated sequences. However, we found that when fragments of radiolabeled long repeat DNA were denatured and reassociated with intact long repeat driver DNA, then sheared to 350 basepairs and assayed for thermal stability, the level of mismatch found in the duplexes varied inversely with the length of the starting fragments. This effect was shown to be due directly to the physical size of the molecules involved in reassociation. These results are consistent with, and support a model for, long repeat DNA in which short units of repetition are arranged in precise arrays. The significance of this arrangement of sequence units within long repeat DNA is discussed.


Action of cathepsin D on human beta-lipotropin: a possible source of human "beta-melanotropin".

Highly purified calf brain cathepsin D (EC 3.4.23.5) selectively splits the Leu77-Phe78 and Ala36-Ala37 peptide bonds of human beta-lipotropin. It is suggested that the formation of human "beta-melanotropin" from gamma-lipotropin, and that of gamma-endorphin from beta-endorphin, is due to the action of cathepsin D during isolation procedures.


Semisynthesis of human somatotropin analogs.

Complementation of the natural NH2-terminal 134-amino acid fragment of the reduced, carbamoylmethylated human somatotropin with synthetic analogs of COOH-terminal fragments of 57, 42, or 38 amino acids of reduced, carbamoyl-methylated human somatotropin has been investigated. It was found that synthetic fragments of 57 and 42 amino acids gave recombinants with full growth-promoting activity, whereas attempts to obtain a recombinant with the synthetic 38-amino acid fragment were unsuccessful. The synthesis of two analogs of the COOH-terminal fragment of human somatotropin, human [N]e170, Ala165,182,189] somatotropin-(150-191) and human [Nle170, Ala165,182,189] somatotropin-(154-191), is herein described.


Origins of phage T4 DNA replication as revealed by hybridization to cloned genes.

[3H]Thymidine-labeled progeny DNA was isolated after infection of Escherichia coli with two different bacteriophage T4 mutants. These strands were isolated shortly after the initiation of DNA replication and hybridized to 15 different (EcoRI) T4 restriction fragments cloned in plasmid vectors. Uniformly labeled T4 [32P]DNA extracted from phage particles was cohybridized as a normalizing reference. The results obtained lead to the conclusions that, among the loci tested, initiation occurs predominantly in the area of genes 50-5 and less prominently in the area of genes 25-29. However, our data do not support the idea of initiation in the area of genes 40-43. In contrast, this area displays the least replication among the genes tested.


Processing in vitro of placental peptide hormones by smooth microsomes.

Rough and smooth microsomes were prepared from ascites tumor cells, rat liver, and bovine adrenal cortex. Proteolytic removal of the signal peptide in pre-placental lactogen and asparagine-linked glycosylation of the alpha subunit of chorionic gonadotropin by these fractions were examined in mRNA-dependent lysates from ascites cells. Both processing steps were performed by smooth microsomes, which was unexpected because it has been presumed that only rough microsomes contain components for ribosomal binding. Thus smooth microsomes are apparently capable of interacting with polysomes bearing secretory nascent chains, and cleavage and asparagine-linked glycosylation activities are present in both rough and smooth endoplasmic reticulum.


Role of proline isomerization in folding of ribonuclease A at low temperatures.

In unfolded RNase A there is an interconversion between slow-folding and fast-folding forms (U(S) right harpoon over left harpoon U(F)) that is known to show properties characteristic of proline isomerization in model peptides. Here, we accept the evidence that U(S) molecules contain nonnative proline isomers and we ask about the isomerization of these proline residues during folding. The U(S) right harpoon over left harpoon U(F) reaction in unfolded RNase A is used both to provide data on the kinetics of proline isomerization in the unfolded protein and as the basis of an assay for measuring proline isomerization during folding.The tyrosine-detected folding kinetics at low temperatures have been compared to those of proline isomerization in unfolded RNase A. The comparison is based on the recent observation that the U(S) right harpoon over left harpoon U(F) kinetics are independent of guanidinium chloride concentration, so that they can be extrapolated to low guanidinium chloride concentrations, at which folding takes place. At 0 degrees C the tyrosine-detected folding reaction is 100-fold faster than the conversion of U(S) to U(F) in unfolded RNase A. Consequently, the folding reaction is not rate-limited by proline isomerization as it occurs in unfolded RNase A. An assay is given for proline isomerization during folding. The principle is that native RNase A yields U(F) on unfolding, whereas protein molecules that still contain nonnative proline isomers yield U(S). Unfolding takes place at 0 degrees C, at which proline isomerization is slow compared to unfolding. This assay yields two important results: (i) The kinetics of proline isomerization during folding are substantially faster than in unfolded RNase A-e.g., 40-fold at 0 degrees C. The mechanism of the rate enhancement is unknown. (ii) At low temperatures (0-10 degrees C), and also in the presence of (NH(4))(2)SO(4), the tyrosine-detected folding reaction occurs before proline isomerization and yields a folded intermediate I(N) that is able to bind the specific inhibitor 2'-CMP. The results demonstrate that a folding intermediate is spectrally detectable when folding occurs at low temperatures. They suggest that low temperatures provide suitable conditions for determining the kinetic pathway of folding by characterizing folding intermediates.


Arrangement of coding and intervening sequences of chicken lysozyme gene.

Hybrid phages that contain chicken lysozyme gene sequences have been isolated from a chicken DNA library. Two overlapping clones covering a region of 22 kilobase pairs around this gene have been studied by restriction mapping. Southern hybridization, and electron microscopic analysis of hybrids between lysozyme mRNA and the cloned cellular DNA. Three intervening sequences interrupt the lysozyme structural gene. The cellular gene is at least 3.9 kilobases long, about 6 times the length of the structural gene.


Unusual nucleotide sequences at the 5' end of actin genes in Dictyostelium discoideum.

The nucleotide sequences at the 5' end of one actin cDNA and six actin genomic clones from Dictyostelium have been determined. The amino acid sequences derived from the nucleotide sequences show strong conservation for six of the seven genes relative to the NH2-terminal region of Physarum actin. The region 5' to the AUG initiating codon is greater than 90% A+T residues in all of the genes.


Discontinuous translation of silk fibroin in a reticulocyte cell-free system and in intact silk gland cells.

Silk fibroin mRNA was translated in a rabbit reticulocyte cell-free system. Addition of tRNA from silk glands was essential for complete translation of the fibroin polypeptide. (Mr approximately 400,000). Synthesis of full-sized product took at least 85 min. In addition to full-size product, a large number of smaller polypeptides were observed upon analysis by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Evidence is presented that these smaller polypeptides are growing fibroin chains that transiently accumulate as discrete size classes due to discontinuities in the translation process. These discontinuities, or pauses, occur at specific sites in the fibroin mRNA template. The relative duration of the pauses can be experimentally modulated by changing the source of the supplementary tRNA added to the in vitro system. Silk glands were incubated in organ culture under conditions where essentially exclusive labeling of newly synthesized fibroins was attained. Analysis in sodium dodecyl sulfate gels showed that the labeling pattern of nascent silk fibroins is similar to the pattern observed in the reticulocyte cell-free system. This result suggests that discontinuities or pauses in polypeptide chain elongation also occur in vivo under conditions of organ culture.


In vitro assembly of intermediate filaments from baby hamster kidney (BHK-21) cells.

Intermediate filaments (IF) from baby hamster kidney (BHK-21) cells can be disassembled at low ionic strength and reassembled upon addition of salt. Turbidimetric analyses show that reassembled IF exhibit the light scattering properties of long rods under physiological conditions (5 mM Na+/K+ phosphate, pH 7.2/170 mM NaCl at 21 degrees C). IF weight concentration, determined by centrifugation, is directly proportional to the optical density at 3000 nm. Thus, turbidity can be used as a quantitative assay for IF assembly. Turbidimetric and centrifugation analyses both indicate that IF assembly exhibits a critical protein concentration of 0.05-0.15 mg/ml. Above the critical concentration, IF weight concentration at steady-state is a linear function of the total protein concentration. Negative stain observations at early stages of the assembly process suggest lateral association of protofilaments to form short IF. This lateral association is accompanied by a rapid turbidity increase which is then followed by IF elongation and a slower turbidity increase to plateau. Further purification of IF by low/high-NaCl-induced cycles of disassembly/reassembly results in retention of 54- and 55-kilodalton (decamin) polypeptides. These results constitute a quantitative description of in vitro reassembly of IF from homogeneous cultures of nonkeratinizing cells and establish conditions for further studies on the regulation of IF assembly.


Asymmetric distribution of plasma membrane proteins in mouse L-929 cells.

The distribution of plasma membrane-associated proteins was studied by using latex-filled phagolysosomes prepared from cultured mouse L-929 cells as a model of "inside-out" membrane. Proteins from 131I/lactoperoxidase-labeled phagolysosomes, phagolysosomes derived from 131I/lactoperoxidase-labeled cells, and phagolysosomes prepared from [35S]methionine metabolically labeled cells were analyzed by high-resolution two-dimensional gel electrophoresis. The gel patterns of iodinated proteins showed specific differences in the availability of membrane proteins to lactoperoxidase labeling between inside-out and right-side-out membranes. However, at least two prominent [35S]methionine-labeled proteins of approximately 60,000 and 100,000 daltons were available for iodination at both sides of the membrane. Partial proteolysis of the 100,000-dalton protein revealed that different peptides were iodinated when the iodination was performed on intact cells or on phagolysosomes, consisent with the idea that this protein spans the plasma membrane.


Cell-cycle-associated rearrangement of inverted repeat DNA sequences.

Inverted repeat DNA sequences of Caulobacter crescentus have been isolated, characterized, and cloned in a bacteriophage lambda vector. Both whole populations and individual clones of these sequences were hybridized to restriction endonuclease-generated fragments of chromosomal DNA isolated from cells that were in different stages of the cell cycle. Some inverted repeat DNA sequences were observed to hybridize to different regions of the chromosomal DNA isolated from the morphologically and biochemically distinct swarmer cell and stalked cell populations. These results suggest that the inverted repeat sequences have the capacity to rearrange and thus be located at different sites on the genomes of the different cell types.


Correlation of polypeptide composition with functional events in acetylcholine receptor-enriched membranes from Torpedo californica.

Membrane vesicles containing partially inactivated acetylcholine receptor (AcChoR) channels may produce a full 22Na+ flux response because an excess of channels may exist above the level needed to completely empty the vesicles of ions. Therefore, attempts to use ion flux amplitudes as indicators of AcChoR function may fail due to the presence of these excess AcChoR channels. Random inactivation of variable fractions of AcChoR channels in vesicles by the irreversible binding of the neurotoxin alpha-bungarotoxin provides a tool for assessing the size of the excess receptor population. Using this approach, we predict that the dependence of the flux response on partial inactivation by alpha-bungarotoxin will drastically change if an essential AcChoR component is substantially removed from the membranes. Membranes from which Mr 43,000, Mr 90,000, and other polypeptides had been substantially removed by base extraction exhibited a flux response after random inactivation that was indistinguishable from that of untreated membranes. Therefore, those components which are substantially removed by base extraction do not appear to be essential for AcChoR-mediated ion flux.


Calcineurin: a calcium- and calmodulin-binding protein of the nervous system.

The inhibitory protein that binds calmodulin and thus prevents activation of several Ca2+-dependent enzymes by calmodulin is shown to also bind four Ca2+ per mol of protein with high affinity (Kd less than or equal to 10(-6) M). On the basis of its Ca2+- binding properties and its localization to nervous tissue, the inhibitory protein is now called "calcineurin." Calcineurin is composed of two subunits: calcineurin A (61,000 Mr) which interacts with calmodulin in a Ca2+-dependent fashion, and calcineurin B (15,000 Mr) which binds Ca2+. The interaction of calcineurin A with calcineurin B is independent of Ca2+ or Mg2+. The dual interaction of calcineurin A with two different Ca2+-binding components and the high affinity of calcineurin for Ca2+ suggest a possible role for calcineurin in the regulation of free Ca2+ concentrations in the nervous system. Calcineurin may thereby modulate the release and action of neurotransmitters.


Hyaluronate and invasiveness of the rabbit V2 carcinoma.

We propose that hyaluronate, a major component of extracellular matrices through which cells migrate during embryonic tissue development and in regenerative processes, is also concentrated in the environment through which neoplastic cells invade local host tissues and may facilitate this process. The hyaluronate content of invasive V2 carcinoma grown in rabbit was found to be 3-4 times greater than that of the same tumor grown in the nude mouse, in which it is noninvasive. Moreover, hyaluronate concentrations were most elevated in the connective tissue interface between the tumor mass and the neighboring host tissue in the invasive rabbit tumors. The particular site of tumor implantation in the rabbit or nude mouse did not affect the concentrations of hyaluronate in either the parenchyma or the surrounding connective tissue. Similar values were obtained for neoplasms grown in muscle, which normally contains little hyaluronate, and in subcutaneous tissue, which is relatively rich in this glycosaminoglycan.


Both chains of HLA-DR bind to the membrane with a penultimate hydrophobic region and the heavy chain is phosphorylated at its hydrophilic carboxyl terminus.

The HLA-DR antigen, a complex of two glycoproteins of 29,000 and 34,000 daltons, can be isolated from the membranes of human B-lymphoblastoid cell lines. Extensive proteolysis releases only 5-10% of the antigen, whereas detergent solubilizes all of it. Detergent solubilization after papain proteolysis of membranes produces antigen with chains cleaved near the carboxyl termini. Comparison of these three preparations demonstrates that each chain contains a carboxyl-terminal hydrophilic region that is sensitive to proteolytic degradation and a penultimate hydrophobic region, responsible for membrane binding, that is more resistant to papain. This two-step cleavage of each chain is also observed during the proteolysis of detergent-solubilized HLA-DR antigen. Both chains of HLA-DR in the membrane can be labeled with the lipophilic photoactivatable carbene reagent adamantane diazirine. This label is released from both chains during the second cleavage. The heavy chain can be reduced and alkylated under mild conditions, and this label is also lost during the second cleavage. The heavy chain is phosphorylated in vivo, and this label is lost upon the first cleavage. This observation suggests that the carboxyl terminus of the heavy chain is intracellular. Cumulatively, these data suggest that both chains of HLA-DR antigens are comprised of large extracellular NH2-terminal regions, small penultimate intramembranous regions, and small carboxyl-terminal intracellular regions.


Changes in activity of fructose-1,6-bisphosphate aldolase in livers of fasted rabbits and accumulation of crossreacting immune material.

The activity of fructose-1,6-bisphosphate aldolase (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13) in livers of fasted rabbits decreases to less than one-half the value found in livers of fed rabbits. However, the concentration of aldolase protein in the liver extracts, measured with a specific antibody, remains unchanged. More than twice as much antibody is required to neutralize the aldolase activity in liver extracts from fasted compared with fed rabbits. The results suggest that modification of liver aldolase occurs during fasting, resulting in loss of catalytic activity without loss of immunoreactivity.


Mechanism of acetylcholine release: possible involvement of presynaptic muscarinic receptors in regulation of acetylcholine release and protein phosphorylation.

Acetylcholine (AcCho) release from purely cholinergic Torpedo synaptosomes was evoked by K+ depolarization in the presence of Ca2+. Activation of muscarinic receptors, present in the synaptosomal fraction, by the agonist oxotremorine resulted in the inhibition of AcCho liberation. This inhibition was abolished by the muscarinic antagonist atropine, which by itself has no effect. These findings suggest that the muscarinic receptor, present in the electric organ of Torpedo is presynaptic and that its physiological function is to regulate AcCho release by negative feedback. The mechanism of presynaptic muscarinic inhibition was investigated by examining the effect of muscarinic ligands on synaptosomal 45Ca2+ uptake and on the level of phosphorylation of specific synaptosomal proteins. Ca2+-dependent K+ depolarization-induced synaptosomal AcCho release was accompanied by 45Ca2+ uptake and by a marked increase in the phosphorylation of a specific synaptosomal protein (band alpha) of approximately 100,000 daltons. Activation of the muscarinic receptor by the agonist oxotremorine had no detectable effect on synaptosomal 45Ca2+ uptake but resulted in the concomitant inhibition of AcCho release and of phosphorylation of band alpha. The muscarinic antagonist atropine abolished the inhibitory effect of oxotremorine both on AcCho liberation and on phosphorylation of band alpha. These findings suggest that phosphorylation of band alpha may be involved in regulation of the presynaptic processes that underly AcCho release and that activation of the muscarinic receptor by agonists may inhibit AcCho release by blocking the phosphorylation of band alpha.


Internal motions in DNA.

We have measured the 31P and 1H NMR parameters of fractionated double-stranded DNA fragments 600, 300, and 150 base pairs long. The results indicate that, inside an intact double helix, both the deoxyribose and the sugar-phosphate backbone of the helix fluctuate substantially from their equilibrium geometry. The time constant for those coupled motions is of the order of 1 nsec.


Enucleation of differentiated murine erythroleukemia cells in culture.

Friend murine leukemia cells induced to undergo erythrocytic differentiation by dimethyl sulfoxide give rise to progeny resembling ortho- or polychromatic normoblasts, which usually do not complete the maturation process to yield forms analogous to erythrocytes. Treatment of these differentiated cells with cytochalasin B can lead to a high proportion (i.e., 80-85%) of enucleated cells in vitro. Nuclear extrusion in cells induced to differentiate by dimethyl sulfoxide and subsequently treated with cytochalasin B began within 24-36 hr of exposure to the antibiotic, with the appearance of a pre-enucleated stage in which the cell nucleus became pycnotic and eccentrically located. Maximum enucleation occurred after 72-96 hr of exposure to cytochalasin B and was significantly enhanced when dimethyl sulfoxide was included in the culture medium during the period of treatment with cytochalasin B. Enucleation appeared to take place only in differentiated progeny, because nondifferentiated cells treated with cytochalasin B alone yielded a population of multinucleated cells. The findings indicate that highly tumorigenic nondifferentiated Friend erythroleukemia cells can be converted in high yield to mature enucleated forms that are unable to proliferate in vitro.


Disappearance of a structural chromatin protein A24 in mitosis: implications for molecular basis of chromatin condensation.

A chromatin protein, A24, a conjugate of histone H2A and evolutionally conserved ubiquitin, was virtually the only structural polypeptide that was present in interphase but missing in mitosis of a Chinese hamster cell line (DON). Because a 10% increase in the H2A/DNA ratio observed in interphase-mitosis transition explained the stoichiometric conversion of A24 to H2A, it appears that ubiquitin bound to H2A of nucleosomal surfaces in interphase is released at mitosis whereas the total H2A remains as a structural component of nucleosomes. Regardless of protein synthesis, ubiquitin was again bound to H2A when cells entered the G1 phase. Based on the electrostatic nature of the COOH-terminal region of H2A, where ubiquitin binds, and the mitosis-specific rise of covalently linked phosphates in histones H1 and H3, we propose that an ionic interaction between the positively charged H2A COOH-terminal regions on fibers and negatively charged phosphates linked to serine or threonine of H1 and H3 molecules on adjacent fibers could generate an assembly of chromatin fibers in mitosis.


Absence of filipin-sterol complexes from large coated pits on the surface of culture cells.

Monolayer cultures of normal or transformed fibroblasts and of liver cells fixed in a glutaraldehyde solution containing 300 microM filipin, a sterol-specific polyene antibiotic, were freeze-fractured to study the distribution of cholesterol within their plasma membranes. Filipin-sterol complexes, recognizable as 25- to 30-nm protuberances scattered in the fracture face of plasma membrane, were absent from invaginations corresponding to large, bristle-coated pits (and possibly also from small, flask-shaped invaginations). These results suggest that invaginating regions on the cell surface are specialized plasma membrane domains with a lower cholesterol content than the surrounding membrane. The localized change in membrane fluidity due to the low cholesterol concentration could play a role in endocytosis.


Cellular regulation of hemoglobin switching: evidence for inverse relationship between fetal hemoglobin synthesis and degree of maturity of human erythroid cells.

To investigate whether the level of maturity of human erythroid cells influences the expression of the fetal hemoglobin program, we studied the relative production of fetal (Hb F) and adult (Hb A) hemoglobins during the maturation of erythroid clones produced in vitro by adult or neonatal erythroid stem cells. In both the adult and the neonatal cell cultures, clones composed of immature erythroblasts showed a significantly higher Hb F/Hb A ratio compared to the mature clones. Culture conditions enhancing erythroid cell maturity (such as an increase in the level of erythropoietin or culture time) decreased the relative synthesis of Hb F in the maturing erythroid cells. Direct immunofluorescence studies demonstrated earlier production of Hb F compared with Hb A during maturation of adult-origin HbF-synthesizing clones. The findings show that the final expression of Hb F is influenced by the degree of maturity of the terminally differentiated cells and suggest that, in addition to regulation at the level of erythroid stem cells, there is control of Hb F expression during erythroblast maturation. The inverse relationship between Hb F expression and level of cell maturity suggests that a regulatory mechanism operating throughout the process of erythroid stem cell differentiation/erythroblast maturation decreases the potential of Hb F expression as the development of the erythroid cell advances.


Tetrodotoxin-sensitive sodium channels in normal human fibroblasts and normal human glia-like cells.

Tetrodotoxin-sensitive sodium channels are detectable in normal human fibroblasts and in "glia-like" cells at appreciable levels when compared to what is observed in established neuronal cell lines in culture. Two- to 3-fold stimulations of sodium influx are observed in the presence of 0.2 mM veratridine and scorpion venom at 0.1 mg/ml. Tetrodotoxin (2 microM) inhibits the observed stimulation of sodium influx. Previous work has indicated that these neurotoxins act on the voltage-sensitive sodium ionophore of excitable cells, and the presence of such channels in cells generally considered nonexcitable raises questions regarding both the uniqueness of this ionophore as a property of excitable cells and the origin of the cells generally described as fibroblasts.


Cytoskeleton-disrupting drugs enhance effect of growth factors and hormones on initiation of DNA synthesis.

Addition of growth factors, such as prostaglandin F2 alpha or fibroblastic growth factor, to quiescent Swiss mouse 3T3 cells resulted in an abrupt increase in the rate of initiation of DNA synthesis after a lag phase of 13-15 hr. This increase could be quantified by a rate constant k. Addition of colchicine, Colcemid, or vinblastine had a synergistic effect on the initiation of DNA synthesis triggered by PGF2 alpha or FGF by increasing the value of k. These drugs alone had no effect. Colchicine had a synergistic effect only if added within 8 hr of the PGF2 alpha or FGF addition. Also, colchicine exerted its full effect when it was present only for the first 5 hr with either growth factor. These results suggest that an intact cytoskeleton is not required for the initiation of DNA synthesis. Furthermore, cytoskeleton-disrupting drugs enhance the stimulatory effect of the growth factors.


Uncouplers of oxidative phosphorylation promote derepression of the hexose transport system in cultures of hamster cells.

Long-term (18-24 hr) preincubation of NIL hamster cell cultures with D-glucose or D-glucosamine (both of which repress the hexose transport system) gave rise to a striking loss of the hexose transport system ("super-repression") when cycloheximide was also present in the culture medium. However, if 0.2 mM 2,4-dinitrophenol (DNP) was also present, the cycloheximide-mediated super-repression was prevented. Moreover, the presence of DNP at this low concentration contributed to an increase in hexose uptake such that it was substantially higher than that permitted by either of the two repressive sugars alone. When the cultures were maintained in medium containing D-fructose in place of glucose, a marked increase in uptake occurred, and this increase (derepression) was not affected by DNP. The derepression due to glucose deprivation and the increases caused by DNP treatment were also observed when 3-O-methylglucose was used to measure hexose transport. Although cultures maintained in the presence of glucosamine exhibited a repressed hexose transport rate, they did not generate significant amounts of lactic acid. DNP, and other uncouplers of oxidative phosphorylation, promoted a derepressed state of hexose transport but did not stimulate the generation of lactate from glucosamine. These data suggest that the metabolic repression phenomena of hexose transport do not depend on glycolysis but rather on the "energized" state of the cell. The energized state of the cell may also be required for the super-repression of hexose transport that is especially apparent when protein synthesis is blocked by cycloheximide.


Lysosomal composition in cultured vascular smooth muscle cells: electron probe analysis.

Spherical electron-dense organelles in the perinuclear region of cultured guinea pig aortic smooth muscle cells were identified as lysosomes by their ability to accumulate acridine orange and by cytochemical demonstration of their acid phosphatase content. The number and size of lysosomes increased in subcultured cells. The elemental composition of the lysosomes was quantitated by electron probe analysis of whole freeze-dried cells and of cryosections. In lysosomes at this stage in their development, the sulfur concentration was higher than that in the cytoplasm and the K/Na concentration ratio was similar to that in the cytoplasm.


Glucocorticoid modulation of casein gene transcription in mouse mammary gland.

The influence of cortisol and prolactin on casein gene expression in the mammary gland of lactating BALB/c mice was measured by using a specific cDNA probe to 15S casein mRNA (cDNAcsn). Casein mRNA (mRNAcsn) level in the mammary gland was decreased by 85% 5 days after adrenal ablation, but then was increased 4.4-fold 12 hr after a single injection of hydrocortisone-21-acetate. An 80% decrease in serum prolactin level, induced by the prolactin inhibitor 2-bromo-alpha-ergocryptin (CB-154), did not alter the level of mRNAcsn in the gland. Specific transcription of the casein gene in nuclei isolated from lactating mammary glands was measured by cDNAcsn hybridization to the in vitro synthesized Hg-CTP-containing RNA (Hg-RNA), which was purified by SH-agarose chromatography. The level of the mRNAcsn in Hg-RNA synthesized in the isolated nuclei was 0.09% and this was decreased 85% by alpha-amanitin, indicating that the mRNAcsn sequences in the Hg-RNA were the products of RNA polymerase II-directed DNA-dependent RNA synthesis. Transcription of the mRNAcsn in isolated nuclei was decreased by 70% 5 days after adrenalectomy and a single injection of the glucocorticoid then increased the transcription level 2-fold at 6 hr. Essentially no alteration of the level of transcription was detectable in mammary nuclei isolated from lactating mice with 80% decreased serum prolactin level, induced by CB-154 treatment. The results thus demonstrate a glucocorticoid involvement on the modulation of casein gene expression at the transcriptional level of control.


Proteases in cellular slime mold development: evidence for their involvement.

Protein degradation appears to be essential for normal differentiation in the cellular slime mold Dictyostelium discoideum. Several protease inhibitors block normal differentiation, and in most cases this inhibition can be reversed by addition of amino acids. For example, chloroquine, which inhibits slime mold cathepsin B activity, interferred with development by blocking sorocarp formation, and this inhibition was reversed by the addition of amino acids. Tosyllysyl chloromethyl ketone also blocked development, and this inhibition was reversed by simultaneous additions of amino acids and glutathione. Moreover, the addition of antipain and leupeptin delayed sorocarp formation. These results, together with the finding reported earlier that cathepsin B activity is differentially localized in the prestalk-prespore zones of the migrating slugs, suggest that proteolysis might play a regulatory role in cellular slime mold differentiation.


Genetic and biochemical analysis of the a1 cell-surface antigen associated with human chromosome 11.

A highly versatile system for genetic, biochemical, and immunological analysis of human cell surface components has been developed using a human-Chinese hamster somatic cell hybrid containing chromosome 11 as its only human chromosome. This system lends itself to studies such as identification of human cell surface antigens and other genetic markers, regional gene mapping of these markers on chromosome 11, mutational analysis of these markers, and exploration of distribution of these antigens in normal and pathological human tissues. Genetic analysis of a1, one of the human antigens expressed by this hybrid, has been accomplished by subjecting a series of a1- variants to complementation analysis. These experiments have shown that the a1- variants behave in a recessive manner and that at least four genes, including three Chinese hamster genes, are needed for a1 antigen expression. Biochemical analysis has shown that a macroglycolipid isolated from human erythrocytes contains the a1 antigenic activity, so that genes coding for glycosyltransferases are required for its biosynthesis and may correspond to the complementation groups identified. The power of combined genetic, biochemical, and immunological approaches to understanding cell membrane molecules is demonstrated.


Human heterozygosity: a new estimate.

Several hundred polypeptides from four human diploid fibroblast cell lines were compared by high-resolution two-dimensional polyacrylamide gel electrophoresis and double-label autoradiography under conditions where allelic products that differ by a single charged amino acid would be distinguished. The average heterozygosity represented by this set of gene products appears to be less than 1% for changes involving charged amino acids.


Characterization of a mutator gene in Chinese hamster ovary cells.

We have recently reported the isolation of a class of mutants (called thy-) that is both resistant to arabinosyl cytosine and auxotrophic for thymidine. thy- mutants have a 5- to 10-fold elevated pool of dCTP and are deficient in the synthesis of dTTP as an apparent consequence of a single mutation in the gene for ribonucleoside-diphosphate reductase (2'-deoxyribonucleoside-diphosphate:oxidized-thioredoxin 2'-oxidoreductase, EC 1.17.4.1). Here we show that three independent thy- lines have a 5- to 50-fold higher frequency and rate of spontaneous mutation for two genetic markers, 6-thioguanine resistance and ouabain resistance. The higher rate of mutation is site specific because two other genetic markers, reversion of proline auxotrophy to proline prototrophy and emetine resistance, are unaffected. Ouabain- and 6-thioguanine-resistant mutations occur at a much lower rate in revertants of thy- to the wild-type state, so the increased rate of mutation is the consequence of the thy- mutation. Both the increased mutational rate and the increased intracellular pools of dCTP are dominant or codominant in hybrid cells, and alterations of the ratio of the pools of dCTP to dTTP in thy- 49 produce corresponding changes in the rate of mutation. Thus, thy- is a mutator gene in Chinese hamster ovary cells, apparently as a consequence of the imbalance of deoxynucleoside triphosphate pools created by the expanded pool of dCTP.


Analysis of genetic complementation by whole-cell microtechniques in fibroblast heterokaryons.

A whole-cell microtechnique for the determination of complementation of human metabolic disorders is presented. This procedure permits the isolation of individual multinucleate cells produced by cell fusion for the quantitative evaluation of complementation. Mutant fibroblasts with a deficiency of propionyl-CoA carboxylase activity (EC 6.4.1.3) that had been mapped to complementation groups pcc and bio were used to evaluate the microtechnique. Complementation was monitored by the determination of [14C]propionate incorporation into cellular macromolecules. Single cells or a small number of cells were isolated from plastic film dishes after radioactive incubation by cutting out the portion of the plastic film holding the desired cells. Isotope incorporation was linear in 10-50 unfused cells and in 10-50 fused normal cells containing five or more nuclei. There was also a direct correlation between the nuclear content of cells and the amount of isotope incorporated. Three pcc and two bio mutants were fused in pairwise combinations by means of polyethylene glycol and complementation was determined by isotope incorporation in sets of 50 multinucleate cells, each cell isolated individually. The results agreed with autoradiographic data for both complementing and noncomplementing strains. The method is quantitative and gives severalfold higher sensitivity than current procedures. The method can be applied to the complementation analysis of a wide variety of inherited disorders of intermediary metabolism.


Partial purification and characterization of DNA from the human X chromosome.

Human X chromosome DNA was partially purified from a mouse-human hybrid cell line containing a single human chromosome, the X. Enrichment of such DNA was accomplished by two sequential reassociations of radiolabeled hybrid cell DNA with large excesses of mouse DNA. Unreassociated hybrid cell DNA was used as a probe for human X chromosome sequences. The human-specific fraction of probe DNA CONTAINED THREE COMPONENTS. Two of these reassociated to human DNAs at rates proportional to the number of X chromosomes present. These two components were thus localized to the X chromosome. One of these X-specific components, representing about 80% of human-specific probe DNA, consisted of single copy or very low order reiterated DNA. The second X-specific component, representing about 10% of human-specific probe DNA, was about 20-30 times more reiterated. The remaining 10% of human-specific probe DNA, although derived from the X chromosome, reassociated to human DNAs at a rate independent of the number of X chromosomes present. This component was thus homologous to autosomal as well as X chromosome DNA. The probe DNA accounts for approximately half of the human X chromosome, suggesting that the remainder may have homology with mouse DNA.


Regulation of mitochondrial malic enzyme synthesis in mouse brain.

In a previous study [Bernstine, E.G. (1979) J. Biol. Chem. 254, 83-87] it was shown that inbred strains of mice fall into two classes based on the specific activity of mitochondrial malic enzyme [L-malate:NADP+ oxidoreductase (oxaloacetate-decarboxylating), EC 1.1.1.40] in brain. In this report we demonstrate differences between high- and low-activity strains in the development of enzyme activity levels in adult mice and show that the rate of enzyme synthesis quantitatively accounts for the inherited level of the brain enzyme. Genetic analysis has established that the locus controlling the amount of enzyme in brain (Mdr-1) is located on chromosome 7. Its linkage to Hbb and c places it in the same region of the chromosome as Mod-2, the structural gene for mitochondrial malic enzyme. By making use of deletions and a duplication that include Mod-2, evidence for cis action of Mdr-1 was obtained.


Evolution of human influenza A viruses in nature: recombination contributes to genetic variation of H1N1 strains.

In June of 1977, a new influenza A pandemic was started by strains of the H1N1 serotype. Oligonucleotide fingerprint analysis of the RNA from viruses isolated during the early stage of this pandemic demonstrated that genetic variation among these 1977 strains could be attributed to sequential mutation [Young, J.F., Desselberger, U. & Palese, P. (1979) Cell, 18, 73-83]. Examination of more recent strains revealed that the H1N1 variants that were isolated in the winter of 1978-1979 differed considerably from the H1N1 viruses isolated the previous year. Oligonucleotide and peptide map analysis of the new prototype strain (A/Cal/10/78) suggested that it arose by recombination. It appears that only the HA, NA, M, and NS genes of this virus are derived from the earlier H1N1 viruses and that the P1, P2, P3, and NP genes most likely originate from an H3N2 parent. These data suggest that genetic variation in influenza virus strains of the same serotype is not restricted to mutation alone, but can also involve recombination (reassortment).


Enhancement of certain biological activities of muramyl dipeptide derivatives after conjugation to a multi-poly(DL-alanine)--poly(L-lysine) carrier.

N-Acetylmuramyl-L-Ala-D-Glu-NH2 (muramyl dipeptide) and several of its derivatives are effective immunoactivators that can enhance nonspecific resistance to infection but can also elicit fever. In contrast, one of its stereoisomers, N-acetylmuramyl-D-Ala-D-Glu-NH2, is devoid of both these activities. Our present report demonstrates that macromolecularization of muramyl dipeptide by attachment of several units to a multi-poly(DL-Ala)-poly(L-Lys) carrier potentiates both its pyrogenic and its immunostimulant activity. This branched polymer has been extensively used as carrier to various haptens. Surprisingly, inactive N-acetylmuramyl-D-Ala-D-Glu-NH2, after conjugation under the same conditions, becomes capable of increasing nonspecific immunity although its lack of pyrogenicity is not greatly modified. Moreover, the N-acetylmuramyl-D-Ala-D-Glu--NH2 conjugate remains devoid of adjuvant, sensitizing, or eliciting activity.


Cell-free synthesis of mouse H-2 histocompatibility antigens.

The mRNAs coding for the histocompatibility (H-2) antigens of the mouse have been identified by cell-free translation of poly(A)-containing RNA obtained from the livers of mice (strain A/J), followed by immunoprecipitation of the cell-free products by using an antiserum directed against purified H-2a. Unlike the 47,000- and 46,000-Mr H-2 glycoproteins synthesized in splenic lymphocytes, the cell-free translation products have Mrs of 45,000 and 44,500, representing the unglycosylated forms of these antigens. The cell-free products are shown to be related to the H-2 antigens by competition immunoprecipitation with purified H-2a and by two-dimensional tryptic peptide mapping. The H-2 mRNAs which sediment at 17 S are found associated predominantly with membrane-bound polysomes and are actively translated in the liver where as many as 16 ribosomes are associated with each molecule of H-2 mRNA. The implications of these studies for molecular cloning and for an understanding of the organization and expression of the genes encoding these H-2 antigens are discussed.


Structural studies of human IgD: isolation by a two-step purification procedure and characterization by chemical and enzymatic fragmentation.

A myeloma IgD immunoglobulin (designated WAH) that was present in high concentration in plasma ( approximately 3.5 g/dl) was purified in >90% yield by a two-step procedure of ammonium sulfate precipitation plus AcA 34 gel filtration. Although the plasma had been stored for 2 years without the addition of a proteolytic inhibitor, no "spontaneous" degradation was apparent and the isolated IgD remained structurally intact. However, the purified IgD showed extreme susceptibility in vitro to various proteolytic enzymes; e.g., Fab(delta) (M(r) approximately 47,000) and Fc(delta) (M(r) approximately 80,000) fragments were generated quantitatively after only 10 min of incubation with papain in the absence of cysteine. By combining limited enzymatic digestion, reductive cleavage, and cyanogen bromide fragmentation, several series of well defined fragments corresponding to the different regions and domains of the IgD molecule were generated. These fragments are useful for physical, chemical, and immunological studies, as well as for the sequence determination of the IgD delta chain. A model of the IgD molecule was derived from such studies and from overlapping of the series of fragments. The possible existence of an extra constant domain in the delta chain appears unlikely in view of our finding of an extended hinge region of about 50 residues which can be cleaved off the amino terminus of the papain Fc(delta) by brief treatment with trypsin. In addition to a distinct stretch of carbohydrate attachment sites, the delta-chain hinge region contains a segment unusually rich in electrical charge. This charged segment is responsible for the lability of IgD to spontaneous degradation and may be related to its biological role as a B lymphocyte receptor.


Modulation of the classical pathway C3 convertase by plasma proteins C4 binding protein and C3b inactivator.

We recently described the isolation from human serum of a serum protein (C4 binding protein) that functions as an essential cofactor for C3b inactivator in the proteolysis of fluid-phase C4b and to a much lesser extent, C3b. We show here the role of C4 binding protein in the formation and function of the classical pathway C3 convertase (C42). C4 binding protein interferes with the assembly of the membrane-bound C3 convertase of the classical pathway and accelerates the decay of C42 in a dose-dependent fashion. Its removal from serum by means of specific immune absorption promotes the vigorous consumption of C3 after addition of C1; this effect is abolished by reconstitution with purified C4 binding protein. Although C4 binding protein inhibits the hemolytic function of cell-bound C4b, we did not detect any change in the structure of C4b even after prolonged incubations of EAC14 with C4 binding protein. For this reason, and on the basis of studies of the time required for maximal reactivity (Tmax) of cellular intermediates generated in the presence of C4 binding protein and limited amounts of C2, we conclude that the effects of C4 binding protein are probably mediated by displacing C2a from specific binding sites on C4b. In addition, C4 binding protein enhances the cleavage by C3b inactivator of the alpha' chain of cell-bound C4b. When EAC14 cells were incubated with both control proteins, the Tmax of the cells was prolonged and the lysis was markedly diminished. We conclude that C4 binding protein and C3b inactivator control the C3 convertase of the classical pathway in a fashion similar to that described for beta 1H and C3b inactivator in the alternative pathway.


Antigenic relationships between measles and canine distemper viruses: comparison of immune response in animals and humans to individual virus-specific polypeptides.

Precipitation with hyperimmune rabbit sera, sera from patients convalescing from measles, and sera from patients with subacute sclerosing panencephalitis, followed by electrophoresis, enabled antigenic relationships between the individual polypeptides of measles and canine distemper viruses to be examined. Virus isolates from patients with acute measles or subacute sclerosing panencephalitis showed no antigenic differences. With rabbit hyperimmune sera, antigenic crossreactivity was present between all polypeptides of measles and canine distemper viruses except H. The N polypeptides showed the highest degree of crossreactivity and were interpreted as group-specific antigens. Both convalescent measles sera and sera from subacute sclerosing panencephalitis showed high antibody titers to all measles polypeptides except L and M. However, these sera contained only low activities to the N and F1 polypeptides from canine distemper virus.


Analysis of human tonsil and cancer DNAs and RNAs for DNA sequences of group C (serotypes 1, 2, 5, and 6) human adenoviruses.

Group C human adenoviruses (Ads) of serotypes 1, 2, 5, and 6 infect most children and commonly cause latent infections of lymphoid tissues. Ads transform cells into a malignant-like phenotype; the oncogenic genetic information is in the left 8% of the viral genome, in the HindIII-G DNA fragment. We have investigated the molecular basis for group C Ad latent infections in human tonsils as well as whether these viruses are linked to human cancer. Tonsil or cancer DNAs and RNAs were assayed for Ad sequences by liquid-phase saturation-hybridization with in vitro-labeled Ad5 HindIII-G fragment. About 25% of the 52 tonsils analyzed contained DNA or RNA sequences specific to HindIII-G, indicating that Ad transforming sequences are expressed as RNA in tonsils. Southern blotting analysis of four tonsil DNAs revealed multiple copies of the complete Ad genome in a free state and provided evidence for an unusual form of the Ad genome, possibly Ad DNA integrated into cellular DNA. In assays of human cancers, no Ad sequences were detected in DNAs from 26 squamous cell carcinomas (Cas), 3 adenocarcinomas, 4 oat cell Cas, 5 stomach Cas, 5 small intestine Cas, 15 colon Cas, 6 rectum Cas, 5 Hodgkin and 6 non-Hodgkin lymphomas, and 2 breast Cas. Reconstruction experiments indicated that the HindIII-G probe could detect 1 copy per cell of 0.2-0.3% of the viral genome. No HindIII-G-specific sequences were detected in RNAs from 21 squamous cell Cas, 3 oat cell Cas, 2 stomach Cas, or 18 colon Cas. In six other experiments using the complete Ad2 genome as probe, no Ad sequences were found in DNAs from 6 lung Cas, 12 normal lung tissues, 33 gastrointestinal Cas, 19 normal gastrointestinal tissues, 6 Hodgkin lymphomas, 3 breast Cas, or 4 kidney Cas, at a sensitivity of about 1 copy per tumor cell of 5-10% of the Ad2 genome. All Ad-induced cancer cells should contain at least 1 copy of 1-6% of the viral genome, the minimal size of the transforming region, and probably should contain multiple copies of more of the genome. Therefore, our data are definitive evidence against group C Ads being the cause of the cancers tested, which represent about 50% of the cancer incidence in the United States. Of additional interest, we did not detect Ad2 sequences in RNAs from 7 human placentas, 12 normal lungs, or 19 normal gastrointestinal tissues (nor in 44 cancer or 23 tonsil RNAs). Thus, we did not confirm a recent report of the presence of Ad2 RNA in RNAs from human placentas; the possibility that a small population of cells in placenta expresses group C "related" sequences is not ruled out.


Tumorigenicity of embryonal carcinoma as an assay to study control of malignancy by the murine blastocyst.

A bioassay, based on tumorigenicity, has been developed to determine the mechanism whereby the blastocyst of the mouse controls malignant expression of embryonal carcinoma. The assay is based upon the incidence of tumors obtained when known numbers of cells of the 402AX strain of embryonal carcinoma are injected into strain 129 mice, compared to the incidence obtained when the same number of embryonal carcinoma cells are incorporated into Swiss-Webster blastocysts that are then injected in strain 129 animals. The results indicate that the blastocyst can regulate one embryonal carcinoma cell consistently; it may have a slight effect on three, but it cannot regulate four or five of them. The position of the embryonal carcinoma cell in the blastocyst is important. Regulation occurs if the embryonal carcinoma cell is placed in the blastocoele cavity, but enhancement of tumorigenicity is obtained if it is placed between the zona pellucida and the trophectoderm. By contrast, the blastocyst is unable to regulate a single B-16 melanoma cell placed in the blastocoele cavity, indicating a degree of specificity for the regulatory process.


Polyamine-pyridoxal Schiff bases in urine.

Schiff bases of the diamines 1,3-diaminopropane, putrescine, and cadaverine and the polyamines spermidine and spermine with pyridoxal or pyridoxal phosphate occur in human urine, as shown by gas chromatographic/mass spectrometric selected ion-monitoring techniques. By use of synthetic standards, procedures were devised for conversion of the Schiff bases to stable derivatives amenable to gas chromatographic/mass spectrometric analysis. These procedures involve borohydride reduction of the C = N double bond, hydrolytic removal of the phosphate group, chromatographic separation from the bulk of urinary constituents, and trifluoroacetylation of polar functional groups. The levels of the polyamine-pyridoxal Schiff bases were estimated to be in the range of pmol/ml or urine.


Purification and primary structure of the neuropeptide egg-laying hormone of Aplysia californica.

Egg-laying hormone (ELH), a neuropeptide synthesized by the bag cell neurons, induces egg laying and its correlated behavior in Aplysia californica. In the present study, ELH has been purified to homogeneity and its primary structure has been determined. We find this molecule to have 36 amino acid residues with a M(r) of 4385 and a calculated isoelectric point of 9.7. Direct microsequence analysis revealed a single amino acid sequence that is in agreement with the amino acid composition determined after acid hydrolysis of ELH: H-Ile-Ser-Ile-Asn-Gln-Asp-Leu-Lys-Ala-Ile-Thr-Asp-Met-Leu-Leu-Thr-Glu-Gln- Ile-Arg-Glu-Arg-Gln-Arg-Tyr-Leu-Ala-Asp-Leu-Arg-Gln-Arg-Leu-Leu-Glu-Lys-OH. Enzyme data indicate that the COOH-terminal lysine may be modified but its exact nature remains to be determined. There is no similarity between the amino acid sequence of ELH and that of presently known vertebrate neuropeptides. The two-step purification procedure, starting with a homogenate of bag cell clusters, consisted of cation exchange chromatography on SP C25 (Sephadex) followed by gel filtration on Bio-Gel P-6. Our purification results in a 100-fold enrichment of ELH from bag cell homogenates and a 36% recovery of purified radiolabeled marker ELH. Analysis of purified ELH radiolabeled with [(35)S]methionine or [(3)H]leucine on isoelectric focusing gels and on 8 M urea/sodium dodecyl sulfate gels showed only a single peak containing 90% of the radiolabel. Radiolabeled ELH migrated with a pI of 9.0-9.2 and an apparent M(r) of 3500-5700. ELH retained egg-laying bioactivity when eluted from this segment of the gel. We find that 2.5 nmol of pure ELH consistently induces egg laying at 20 degrees C.


Induction of maternal behavior in virgin rats after intracerebroventricular administration of oxytocin.

Oxytocin produces uterine contractions and milk ejection, functions related to parturition and nuturing. Studies were conducted to determine if this peptide, native to the brain and the posterior pituitary gland, plays a role in the induction of maternal behavior. Intact virgin female rats were given 0.4 mug of oxytocin, 0.4 mug of [Arg(8)]vasopressin, or saline through lateral ventricular cannulae. Forty-two percent of intact rats receiving oxytocin displayed full maternal behavior towards foster pups. None of the saline- or vasopressin-treated animals displayed full maternal behavior. Criteria in five behavioral categories had to be fulfilled by an animal within 2 hr of injection for its behavior to be considered fully maternal. When partial maternal responses were considered, oxytocin was significantly more effective than saline and marginally more effective than vasopressin. Five animals responding fully maternally after oxytocin injection were allowed to stay with pups for 10 days. All five continued to display full maternal behavior during this time. Nearly all animals that responded fully maternally to oxytocin injection were in the last day of diestrus or in proestrus or estrus. This suggested that elevated or recently elevated levels of estrogen may be necessary for the induction of full maternal behavior by oxytocin. Twenty-seven virgin female rats were ovariectomized and given either 100 mug of estradiol benzoate per kg in oil subcutaneously or oil alone immediately after operation. Forty-eight hours later, all animals received 0.4 mug of oxytocin intracerebroventricularly. Eleven of 13 estrogen-primed animals became fully maternal; none of 14 nonprimed animals became fully maternal.


Classical conditioning in Aplysia californica.

A form of aversive classical conditioning is described in which a chemosensory conditioned stimulus rapidly acquires the ability to modulate a defensive response (escape locomotion). Because Aplysia show both sensitization and classical conditioning, it is now possible to begin to examine the relationship between nonassociative and associative learning on behavioral and cellular levels.