Panel discussion: glomus jugulare tumors of the temporal bone. Radiological appearance of glomus tumors. Depending upon the stage of pathogenesis, some glomus tumors demonstrate characteristic radiographic findings which permit a presumptive diagnosis of this type of tumor. Three factors are helpful in interpreting multidirectional tomography of the temporal bone when a glomus tumor is suspected. The tumor shows a predilection for bony erosion of the carotid crest and hypotympanum. It tends to destroy bone in its path by small lobulated invasions producing a fairly well demarcated border of crescentic shaped translucencies. In contradistinction to other tumors and infections, glomus tumors tend to spare the lateral wall of the attic and the ossicular chain even when the tumor if fairly well advanced in size.


Panel discussion: glomus jugulare tumors of the temporal bone. Radiation of glomus tumors of the temporal bone. In a study done at the Geisinger Medical Center in 1976, the results of treatment of 30 cases of glomus tumors involving the ear were reviewed. All 8 cases of glomus tympanicum were controlled with surgery and all of the cases of glomus jugulare treated with high voltage radiotherapy were controlled for from 2 to 12 years. Currently, the length of control extends to 15 years and there have been no late recurrences. Three additional cases showing unusual features of glomus jugulare are presented.


Panel discussion: glomus jugulare tumors of the temporal bone. Patterns of invasion in the temporal bone. The purpose of this paper is to enumerate and define the exact patterns of glomus jugulare tumor invasion in the temporal bone. Special attention will be focused on those pathways which lead to central nervous system (CNS) invasion.


Panel discussion: glomus jugulare tumors of the temporal bone. The surgical management of glomus tumors. The authors describe the diagnosis and surgical management of 70 glomus tumors seen at the Otology Group in an 8 yr. period. The need for bilateral arteriograms to detect associated tumors (carotid body and intervagale) is stressed. A new surgical technique, the modified intratemporal fossa approach, is described for the removal of large glomus jugulare tumors involving the carotid artery. Complications and results are discussed in detail.


Congenital cytomegalovirus and hearing loss: clinical and experimental observations. The effects of congenital cytomegalovirus infection vary from severe central nervous system destruction to asymptomatic viuria. Yet children with the latter have a high incidence of unsuspected hearing loss. Histopathology of temporal bones from children who die with cytomegalic inclusion disease shows viral infected cells in non-sensory endolabyrinthine epithelium. Experimental mouse cytomegalovirus causes an age related cranial neuronitis and perilabyrinthitis. Immunopathologic localization of different viral antigens in experimental inner ear infections shows differential vulnerability of labyrinthine structures to infections. Adaptation of such techniques to human temporal bone pathology could yield information regarding pathogenesis of viral labyrinthitis. The development of a human cytomegalovirus vaccine may eliminate the risks of this congenital infection.


Autogenous vaccine treatment of juvenile laryngeal papillomatosis. Seventeen cases of juvenile laryngeal papillomatosis have been seen and treated with microlaryngoscopy, removal of papillomas, and administering of autogenous vaccine. Holinger's original findings could be confirmed. The operation frequency of 9 patients was significantly reduced, 5 were improved, and 3 unchanged. In no case was an undesirable reaction to the vaccine observed. Electron microscopy showed no virus-like particles in the papilloma but a section of a vulvar wart did show the virus.


Fibrous histiocytoma of the parotid gland. Fibrous histiocytomas are not uncommonly found in the head and neck region. These tumors, arising from the tissue histiocyte, generally behave in a benign fashion. An unusual case of a fibrous histiocytoma arising in the parotid gland is presented. The histopathology of these lesions as well as parotid neoplasms in children is reviewed. Wide local excision without sacrifice of important structures is the treatment of choice.


[Determination of free crystalline silica in the dust containing silicates]. The content of free crystalline silica in dusts containing silicates was investigated using Polezajew's method, the HBF4 method and X-ray diffraction. Polezajew's method was found to give highly increased results (133.4% on the average), and with 5% of quartz in mixture with kaolin--252%. Solution of silicates, when heated, in fluoroboric acid considerably reduces the error of determination. The mean value of SiO2 recovery in mixtures of quartz with kaolin is 102.9% and with 5% of quartz--116. This method is highly precise. The results of determination of crystalline silica content in some mineral raw materials used in ceramic industry approximated the results obtained using X-ray diffraction.


Preparative scale isolation of basal-lateral plasma membranes from rat intestinal epithelial cells. A simple, efficient procedure is described for the preparative scale isolation of basal-lateral membranes from the rat intestinal epithelium. The intestinal mucosa was mildly homogenized and soluble protein and RNA were separated from the homogenate by differential centrifugation. The basal-lateral membranes were then separated from nuclei, mitochondria, and brush border membranes by differential centrifugation in a medium close to the equilibrium density of the basal-lateral membranes. Final purification of the basal-lateral membranes was achieved on a linear density gradient in a high-capacity zonal rotor. The final product (usually at least 40 mg protein) represented a 34% yield of basal-lateral membranes purified 18-fold with respect to protein, 26-fold with respect to brush border membranes, and 53-fold with respect to mitochondria.


Stability of rhodopsin in detergent solutions. The thermal stability of lipid-free rhodopsin in solutions of a homologous series of alkyltrimethylammonium bromide detergents and one nonionic detergent, dodecyl-beta-maltoside, has been studied as a function of detergent concentration. Rhodopsin thermal stability increases with increasing chain length within the homologous series of ionic detergents, and for chain lengths greater than 10 carbon atoms increases with increasing detergent concentration up to a "critical" concentration that depends on the chain length. Stability also increases with increasing detergent concentration for rhodopsin in solutions of the nonionic detergent. These results may be rationalized in terms of the dependence of micelle packing density on the detergent chain length, head group, and concentration.


Plasma concentrations of unconjugated and conjugated estrogens and gonadotrophins following application of various estrogen preparations after oophorectomy and in the menopause. In 55 patients after oophorectomy and 20 women after natural menopause an oral estrogen replacement therapy was performed with estrone-sulfate, estradiol 17-valerate, estriol-succinate, a combination of micronized estradiol and estriol (Estrifam, Trisequens), and natural conjugated estrogens. In 4 patients a 3 mg estradiol per 5 g ointment substance was applied on the abdominal skin. The interindividual variations of estrogen increments during therapy were considerably high. Oral intake of 2 mg estriol-succinate daily was followed by a 500% increase of total (conjugated + unconjugated) estriol. Concentrations of unconjugated estrogens were not altered by this dosage. Following oral application of the other above mentioned preparations, prominent rises--especially of unconjugated estrogens in plasma--were noted. The concentration peaks occurred within 3--6 h after application. Unconjugated estradiol-17 beta in plasma was comparable with values of the follicular phase of a normal menstrual cycle, unconjugated estrone, however, was nonphysiologically high. Consequently, the E1/E2 ratio was greater than one whereas it is normally below one. 12 h after oral estrogen application, plasma estrogens dropped to almost initial values, so that a second medication seems to be necessary in order to guarantee an adequate supplementation over the course of the day. The hormone values determined in this study did not show significant differences between patients after a natural menopause and after oophorectomy. There was a positive correlation between rising estrogen levels and suppressed gonadotrophins during replacement therapy. The occurrence of climacteric symptoms did not exclusively depend on low estrogen and high gonadotrophin levels. Good tolerance of estrogen therapy with significantly elevated estrogen concentrations in plasma can be obtained transcutaneously in the form of estrogen ointments. Such therapy might simulate the physiological estrogen pattern even better than oral application does because of delayed and diluted steroid flow to the liver.


Seroconversion to human-cytomegalovirus-specific pre-early nuclear antigens in renal transplant recipients. Seroconversion to human cytomegalovirus (H-CMV)-specific antigens was observed in 8 out of 9 renal transplant recipients. In 7 of the 8 recipients antibody to "pre-early nuclear antigens" (PENA), which are detectable in human embryonic lung cells within 1 hr of H-CMV infection by anti-complement immunofluorescence staining, developed concomitantly with the increase in other antibodies including anti-early antigens (EA), anti-nuclear inclusions (NI), and complement-fixing (CF) antibody in 1--2 months after transplantation. About 1 year later, anti-PENA and anti-EA titers were concomitantly decreased in 2 recipients, whereas anti-NI and CF antibody titers were maintained at elevated levels in all the seroconverted recipients. These results support the idea that the development of antibody to PENA, like antibody to EA, may represent a current or recent infection with (or reactivation of) H-CMV. In one patient, antibody to PENA did not develop through the observation period despite increases in antibody to EA and other antibodies; this lends support to immunological distinctness of PENA from EA.


Interaction between 30 S ribosomal components in a viomycin resistant mutant of Mycobacterium smegmatis. A high level viomycin resistant mutant of Mycobacterium smegmatis ATCC 14468 (AC16) was analyzed genetically and biochemically in an attempt to understand the mechanisms of expression of high level viomycin resistance and co-resistance to kanamycin and streptomycin. Genetic analysis has shown that at least three different genes (vicC, str, and kan) were involved in the phenotypic expression of drug resistance in AC16, and high level resistance to viomycin was due to interactions between the products of these genes.


Characterization of bovine parainfluenza virus type 3. Bovine parainfluenza virus type 3 (PIV-3) has a buoyant density of 1.197. The RNA of PIV-3, like that of Sendai virus, is a single continuous chain which lacks polyadenylic acid sequences and tends to self-anneal to a marked extent. It has a sedimentation coefficient of 42S and a molecular weight of 4.5 X 10(6), being slightly smaller than Sendai virus RNA (47S, 5.3 X 10(6)). PIV-3 has 5 main structural proteins, of which 2 are glycoproteins. The molecular weights of protein 1, protein 2, protein 3, glycoprotein 1, and glycoprotein 2 were estimated to be 79,000, 68,000, 35,000, 69,000, and 55,000, respectively. Protein 2 was suggested to be nucleocapsid protein.


Enrichment of C-type virus mRNA from immunochemically separated polyribosomes. A method for the isolation of mRNA from a c-type virus-positive cell (JLS-V6) has been developed. This method consisted of (i) specific immunochemical precipitation of polyribosomes synthesizing viral proteins, (ii) extraction of the mRNA from the polyribosome/antigen/antibody complex, and (iii) separation of poly-A containing RNA by affinity chromatography on oligo-d(T)-cellulose. Three size classes of virus specific poly-A containing mRNA are identified and they are 35S, 20S-25S, and 10S-15S.


Effect of nucleosides on interferon production and development of antiviral state induced by poly I.poly C. Effect of nucleosides both on induction of antiviral state in chick embryo cells (CEC) or rabbit kidney cells (RK13) and on interferon production in RK13 or mouse fibroblast cells (L cells) by polyriboinosinic-polyribocytidylic acid (poly I.poly C) was studied. Addition of inosine or a fifty-fifty mixture of inosine and uridine at a final concentration of 0.1 mM to 10 mM to a growth medium enhanced development of antiviral state in CEC. The nucleoside effect was also observed in RK13 at 0.1 mM but not at a concentration higher than 1 mM. Interferon production in RK13 by superinduction (sequential treatment with metabolic inhibitors after exposure to poly I.poly C) was enhanced 1.5- to 4.0-fold by addition of the nucleoside mixture to the growth medium. When RK13 was pretreated with 10 units per ml of interferon and then superinduced by inhibitors, the enhancing effect of nucleosides on interferon production was not observed. Interferon production in L cells was potentiated a little by addition of 1 mM of the nucleoside mixture to the growth medium. The effect of nucleoside was not observed when the nucleosides were added after exposure to poly I.poly C. The nucleoside effect may be applicable for production of high titered interferon.


Reactivity of neutralizing antibodies with different specificities to H particles of poliovirus. In order to elucidate the antigenic structure of poliovirus, the reactivity of antibody produced with H antigenic particles of Mahoney strain (polio type 1) was investigated. Injection of H particles of Mahoney strain into rabbits yielded neutralizing antibody as well as CF-N and CF-H antibodies. This result coincided with the report by Hinuma and coworkers. Neutralization tests with inhibitor resistant Mahoney mutants revealed that the neutralizing antibody produced with H particles was of HN31 type, one of the five different kinds of polio neutralizing antibodies reported previously (14). Absorption experiments with H particles on different neutralizing antibodies and analysis of antibody eluted by acid dissociation from antiserum-treated H particles also showed that the HN31 type antibody specifically combined with H particles of Mahoney strain. Since the H particle of poliovirus is known to be deficient in VP4, these results seems to indicate that the HN31 type antibody reacts with a structural part(s) of poliovirus other than VP4.


Bilateral Wilms tumor presenting with acute renal failure and clinical findings mimicking cardiac failure. A six-year-old girl presented with clinical signs and symptoms of right-sided heart failure. Cardiac catheterization demonstrated filling defects in both pulmonary arteries felt to represent metastatic lesions or thromboemboli. Intravenous pyelogram revealed bilateral renal masses and intrarenal obstruction. Acute renal failure was unresponsive to standard therapy. An open renal biopsy revealed Wilms tumor with favorable histology. Aggressive chemotherapy and irradiation were undertaken and renal function returned. This case adds to the heterogeneity of presentations described for Wilms tumor, and review of the literature reveals no other cases with similar presenting signs.


[Possible pathways for acetyl-CoA formation by purple bacteria]. Purple sulfur (Ectothiorhodospira shaposhnikovii, Chromatium minutissimum, Lamprobacter modestohalophilus, Thiocapsa roseopersicina) and nonsulfur (Rhodospirillum rubrum, Rhodopseudomonas palustris, Rhodopseudomonas spheroides) bacteria are capable of forming acetyl-CoA synthetase, phosphotransacetylase and acetokinase independent of the medium composition and growth conditions. In all of the purple sulfur bacteria with an exception of E. shaposhnikovii, the activity of acetokinase is much higher than in purple nonsulfur bacteria. Apart from being involved in the synthesis of acetyl-CoA, such enzymes as phosphotransacetylase, acetokinase and adenylate kinase may play an important role in energy processes of some purple bacteria in the dark.


Oxidative inactivation of the molybdenum-iron-protein component of nitrogenase from clostridium pasteurianum. The sensitivity of the molybdenum-iron(MoFe)-protein of Clostridium pasteurianum nitrogenase toward oxidation has been studied by determining the enzymatic activity of this component after incubating it anaerobically in ferricyanide solutions of various oxidizing strengths (as measured by their oxidation potentials). It was found that the MoFe-protein remains active at potentials up to +350 mV (vs. standard hydrogen electrode) but becomes readily inactivated at more oxidizing potentials, after a lag period, depending on the potential level and temperature. Oxidative inactivation by ferricyanide results in the release of most of the Mo, Fe and S atoms from the protein which causes the loss of the absorption bands in the visible region. The metals and sulfur could be re-incorporated by incubation in a mixture containing thiol, sulfide, molybdate, and ferric iron. The EPR spectrum of the oxidatively inactivated MoFe-protein showed that both the high- and low-field signals are readily affected. Re-incorporation of the metals and sulfur into the "bleached" protein produced an EPR spectrum similar to that of the air-inactivated protein. Incubation of the Mo-Fe-protein with mersalyl abolished its enzymic activity. The difference spectrum before and after mersalyl treatment resembles that of the soluble spinach ferredoxin.


Properties of protease-treated cytochrome c oxidase from beef heart. About 45% of the protein can be removed from oxidized cytochrome c oxidase by treatment with proteolytic enzymes under a variety of conditions, leading to an increased heme to protein ratio. The principal spectroscopic parameters of cytochrome c oxidase are retained in the protease-treated enzyme. Of the overall catalytic activity 20% remained after digestion; the electron-transfer reactions were impaired but the affinity for cytochrome c appeared unchanged. Proteolysis resulted in removal of the hydrophobic subunit III and most of the smaller hydrophilic subunits, leaving a core, which basically consists of the two largest subunits I and II. The subunits I and/or II carry the prosthetic groups of the enzyme and at least one of the cytochrome c binding sites. The smaller subunits, however, are essential for optimal electron transfer and possibly have other functions as well.


The time course of the phosphorylation of proteins in the synaptic plasma membrane and the effect of certain cations. The phosphorylation of proteins in the synaptic plasma membrane is a rather slow reaction taking several minutes to saturate all the phosphate acceptor sites. (The time for half the protein bound phosphate groups to turnover is about 1 min). A divalent cation is needed as a cofactor for the reaction. At high (0.5 mM) ATP concentrations Mg2+ is more effective than Mn2+ but at low (10 microM) ATP concentrations the reverse is the case. Zn2+ and Ca2+ support very little phosphorylation.


Characterisation of poly(ADP-Rib) polymerase activity in nuclei from the slime mould Dictyostelium discoideum. Nuclei isolated from D. discoideum and incubated in vitro with 3H-NAD synthesise poly(ADP-Rib). The optimum incubation conditions for the poly(ADP-Rib) polymerase were determined. The Km of the enzyme is 18 microM NAD and it is inhibited by nicotinamide. Most of the poly(ADP-Rib) synthesised is attached to nuclear proteins.


[DNA fragmentation of chicken adenovirus CELO by specific endonucleases R. HpaI, R. EcoRI, R. HindIII]. The effect of specific endonucleases on DNA chiken adenovirus CELO was studed. It was shown that endonucleases R. HpaI, R. EcoRI and R. HindIII cleaved viral DNA into 5,7 and 9 specific fragments, respectively. The sequence of fragments (physical map) was determined and found to be: D-A-E-C-B for enzyme R. HpaI; B-(EG)-C-A-D-F for enzyme R. EcoRI; H-F-A-C-G-B-D-E-I for enzyme R. HindIII.


[Electron paramagnetic resonance study of electron transport in photosynthetic systems. VIII. The interplay between two photosystems and kinetics of P700 redox transients under various conditions of flash excitation]. The kinetics P700 redox transients in bean chloroplasts induced by short (t 1/2 = 7 microsecond) flashes of white light has been studied. The flashes were fired in the dark or simultaneously with the continuous background far red light (707 nm). After oxidation of electron transport chain by 707 nm light only 50% of P700+ centres were reduced by photosystem 2 in response to the first flash of saturating intensity. The total reduction of P700+ centres was observed only after two or more saturating flashes. The similar data were obtained for various native photosynthetic systems (bean leaves and leaves of other higher plants). It has been shown that the quantitative difference in P700 behaviour after the first and following flashes may be interpreted in terms of a mechanism of the plastoquinol oxidation by photosystem 1. Twofold oxidation of one molecule of plastoquinol was assumed as its successive oxidation to plastosemiquinone and plastoquinone by different acceptors localized nea the same P700 centre (cytochrome f and plastocyanine). It has been shown that one molecule of plastoquinol was arised due to the interaction of two plastosemiquinones reduced by different P680 centre.


Survival and herpes virus production of normal and xeroderma pigmentosum fibroblasts after treatment with formaldehyde. The survival of excision-deficient and of excision-proficient (variant) skin fibroblasts from xeroderma pigmentosum (XP) donors was about 5 times and twice, respectively, more sensitive to formaldehyde (FA) treatment than that of skin fibroblasts from healthy and XP heterozygote donors. The capacity of FA-treated host cells to further support Herpes virus (HSV) replication was also more sensitive to FA in XP12BE (group A) than in normal (KD) cells. An important recovery of this capacity occurred in both cell types when they were infected at increasing times (up to 36 h) after FA treatment. This contrasts with the decreasing capacity observed in XP12BE when similarly infected at increasing times after exposure to ultraviolet. In addition, the survival of FA-treated HSV was comparable in KD and XP12BE cells, whereas that of UV-irradiated HSV was much lower in XP12BE than in KD cells.


[Myxovirus structural investigations by spin probe methods. II. Influence of rimantadine on the structure of viral and artificial lipid membranes]. The lipid membrane structure of two myxoviruses (influenza and Sendai viruses), red blood cells and liposomes (prepared from total lipids of Sendai virus) have been investigated in the presence of rimantadine by means of two spin probes C5 and C6--the iminoxyl derivatives of stearic acids. Rimantadine was shown to penetrate the lipid membranes of myxoviruses and red blood sells and change the structure of the lipid phase no less than 0.8 nm apart from the viral surface. In the depth of 2.2 nm the structure of these lipid membranes remains unchanged after addition of rimantadine. Rimantadine also displaces the phase transition points of cell and viral lipid membranes to lower temperatures. Rimantadine does not change the lipid bilayer structure of liposomes 0.8 nm apart from its surface. These finding allow to suggest that rimantadine affects the cell and viral lipoprotein membranes.


Polyaxonal myelination in developing dystrophic and normal mouse nerves. Myelin-forming Schwann cells in the peripheral nervous system characteristically surround and myelinate only single axons. Polyaxonal myelination is an anomaly of this one-to-one relationship whereby one normal-appearing Schwann cell myelinates multiple axons. We examined the ventral roots and the proximal sciatic and posterior tibial nerves of developing normal mice and of dy2J/dy2J dystrophic mice with proximal failure of myelination. Polyaxonal myelination was a rare feature in normal nerves. Examples of polyaxonal myelination were observed six times more often in dystrophic than in normal mice and were most abundant in proximal sciatic nerves. Polyaxonal myelination could result from either an axonal or a Schwann-cell abnormality, or it may be the nonspecific response of uncommitted Schwann cells to an early failure of myelination.


Effects of D-penicillamine on neuromuscular transmission in rats. Treatment of patients with D-penicillamine (D-P) has been associated with a syndrome similar to myasthenia gravis (MG). To explore this association, we examined the effects of D-P on neuromuscular transmission in rat muscle. In the first experiment, bath-applied D-P had no significant effect on either miniature endplate potential (MEPP) amplitude or action potential (AP) amplitude. Endplate potential (EPP) amplitude and spontaneous MEPP frequency decreased significantly at concentrations approximately 40 times the maximum human therapeutic level. In the second experiment, rats receiving D-P by daily injections for 33 to 37 days did not differ from controls in any of the measured electrophysiologic characteristics. Electron microscopy of muscle endplates from rats treated with D-P showed no evidence of degeneration or simplification. In all cases, thymus histology by light microscopy was normal, and no antireceptor antibodies were found. Thus, D-P has a mild direct presynaptic effect on neuromuscular transmission at high concentrations, but this effect is too small to account for the weakness seen in the myasthenia-like syndrome in humans.


Penicillamine-induced myasthenic responses in the guinea pig. To study the basis for the myasthenic illness that develops in some patients receiving D-penicillamine (D-P), we measured neuromuscular transmission in D-P-treated guinea pigs. Treated animals manifested edrophonium chloride-repairable decremental responses to 30-Hz nerve stimulation (8.5% +/- 1.5%--mean +/- SEM; N = 16) compared with the response in saline-treated control animals (-0.3% +/- 0.85%, N = 10, p less than 0.005). Posttetanic phenomena were studied in 5 treated animals; 3 revealed posttetanic exhaustion. Miniature endplate potential (MEPP) amplitudes were significantly reduced in 11 treated animals (0.612 +/- 0.016 mV) compared with 6 controls (0.713 +/- 0.024 mV, p less than 0.01). The MEPP frequency appeared reduced in treated animals. Both MEPP amplitude and MEEP frequency correlated with the decrement.


Onset of neural function in the lateral line. The development of the nervous system includes the formation of specific neuronal connections. Some insight into the mechanisms by which these connections are made may be obtained by determining the order in which the elements of a developing system begin to function and examining any changes that may occur in the system shortly after it begins functioning. I have investigated the lateral line of the South African clawed frog, Xenopus laevis and I have classified the afferent system into three elements. First, the transduction mechanism, which includes all components that couple the water vibrations to the release of transmitter from the hair cell. Second, the afferent synapse, at which there is transmitter release and subsequent postsynaptic conductance changes. And third, the afferent axon. I report here experiments suggesting that these elements become functional in the following order: first, the axon can conduct action potentials; second, transmission is established; third, the transduction mechanism can modulate transmitter release. This same sequence has been reported in a system related to the lateral line, the auditory pathway of mammals.


Gonadotropin-releasing hormone analogue binds to luteal cells and inhibits progesterone production. Although gonadotropin-releasing hormone (GnRH) is believed to mediate the hypothalamic control of pituitary gonadotropin secretion, continuous or repeated administration of GnRH or its agonist analogues has been shown to cause paradoxical antifertility effects in several species, including primates. GnRH-induced interruption of reproductive cycles and pregnancy is associated with diminished progesterone production, implying defective function of the corpus luteum. These luteolytic effects have been attributed to the well recognized desensitising actions of elevated luteinising hormone (LH) levels on ovarian LH receptors and steroidogenesis, subsequent to GnRH-induced gonadotropin release from the anterior pituitary. However, treatment with high doses of exogenous LH did not cause suppression of serum progesterone levels during early pregnancy in rats, whereas a highly active GnRH analogue was effective in this regard. These observations suggested that GnRH and its agonist analogues, given in high or sustained doses, can exert a direct action on the ovary that is independent of the pituitary. This hypothesis was further supported by the ability of GnRH and its agonists to inhibit human chorionic gonadotropin (HCG)-induced ovarian and uterine weight gain in hypophysectomised rats and to delay the onset of puberty in intact female rats. Also, GnRH and its agonist analogues have recently been shown to inhibit steroidogenesis induced by follicle-stimulating hormone (FSH) in cultured granulosa cells, confirming the direct action of such peptides on the ovarian follicle. The marked inhibitory effects of GnRH and its agonists on corpus luteum function suggest that these compounds could exert direct actions by binding to specific receptors on luteal cells. The present experiments, which examine the effects of GnRH agonists on luteal steroidogenesis, demonstrate the existence of such actions and their mediation by specific high-affinity receptor sites present in luteal cell membranes.


Phosphorylation of adenosine in aqueous solution by electric discharges. Dehydration reactions involving condensing reagents in aqueous solution have been studied as models of chemical evolution, and one such reagent has been found to be produced in the supposed primitive Earth conditions. We assumed that the dehydration condensation in aqueous solution could occur if the condensing reagents, which appear to be produced by electric discharges in the gas phase, could be carried to the aqueous solution through the recycling of water washing the wall of the vessel. We present here an experimental study of the dehydration condensation between phosphate and adenosine in aqueous solution using a new discharge apparatus (Fig. 1) which simulates prebiotic chemical evolution. The apparatus was designed so that water recycles in a vessel containing a solution at a relatively low temperature (approximately 30 degrees C), achieved by showering water at 18 degrees C from above the vessel. This is a simulation of the recycling system of water on the Earth, depending on a large difference in temperature between the ocean surface and the sky.


Endogenous viral genes are non-essential in the chicken. DNA sequences homologous to the genomes of type C retroviruses are widespread among vertebrates. Ten genetic loci containing endogenous viral DNA sequences have been documented in the white Leghorn chicken alone. Six of these genetic loci are associated with the production of virus or of viral proteins in embryonic fibroblasts (refs 2--4, and S.M.A., L, B. Crittenden and E.G.B., in preparation) and one of the loci may be expressed in the erythroblasts of 5-day-old embryos. The abiquitous presence of endogenous viral genes among vertebrate species and the association of their expression with development of the haematopoietic system in the mouse have led to the proposal that these genes are involved in ontogeny. In addition, the genes may be implicated in oncogenesis as in the case of the AKR mouse in which a high incidence of spontaneous leukaemia is associated with the expression of endogenous murine laukaemia virus genomes. We report here the production of a fertile rooster which lacks avian leukosis virus-related endogenous viral genes and which seems to be completely normal and healthy. Thus, endogenous viral genes are apparently not essential for the normal development of the chicken. An endogenous virus-free state has also been reported for three species of jungle fowl and for the B-type viral genes of the mouse.


A novel analgesic dipeptide from bovine brain is a possible Met-enkephalin releaser. It is generally accepted that morphine exerts its analgesic effect by binding to specific opiate receptors in the brain and spinal cord. Since Hughes et al. isolated and identified two endogenous pentapeptides, Met- and Leu-enkephalin, from the brain and found that they acted as agonists at opiate receptors, alpha-, beta- and gamma-endorphins, larger peptides than enkephalins and having morphine-like activity, have been identified in either the brain or pituitary of various species. Several studies have demonstrated that enkephalins possess analgesic properties and that they are distributed in the pain-mediated pathways in the central nervous system. These findings suggest that enkephalins are important neurotransmitters or neuromodulators regulating pain transmission. We now report the isolation of a novel substance which has a Met-enkephalin releasing action. Our findings suggest the possibility of a regulating mechanism for the release of endogenous opioid peptides, especially Met-enkephalin.


Apamin blocks certain neurotransmitter-induced increases in potassium permeability. Apamin is a neurotoxic polypeptide of known structure isolated from bee venom. Shuba and coworkers have recently shown that it abolishes the hyperpolarising action of externally-applied ATP on visceral smooth muscle (guinea pig stomach and taenia coli) as well as the hyperpolarisation (inhibitory junction potential) that follows stimulation of the non-adrenergic inhibitory nerve supply to these tissues.  As it has been proposed that ATP is the neurotransmitter involved in the latter response, Vladimirova and Shuba tentatively concluded that apamin is a specific postsynaptic blocking agent of this non-adrenergic, possibly 'purinergic', inhibition. We have confirmed the important observation that nanomolar concentrations of apamin reduce inhibition by ATP and by non-adrenergic nerve stimulation, but further experiments suggest that, rather than acting as a specific blocker of ATP receptors, apamin inhibits the increase in potassium permeability caused by a number of agents, including ATP.


Desensitisation of cultured glial cells to epidermal growth factor by receptor down-regulation. Epidermal growth factor (EGF), which can be purified from the mouse submaxillary gland or from pregnant human urine, is a potent multiplication-stimulating factor for several types of cultured cells, including human fibroblasts and glial cells. The molecule binds with high affinity and saturation kinetics to a cell-surface receptor, is subsequently internalised and finally degraded. The binding event is accompanied by a reduction in the number of EGF receptors. This phenomenon--'receptor down-regulation'--has been demonstrated with several hormones and may be a general principle for the modulation of binding groups on the outer cell surface. Further, it has been proposed that receptor loss acts to regulate the cellular response to the binding ligand. The present study provides direct experimental support for this hypothesis. It demonstrates that down-regulation of EGF receptors on glial cells causes desensitisation of the mitogenic response of these cells to subsequent stimulation with EGF.


Enkephalins presynaptically inhibit cholinergic transmission in sympathetic ganglia. Recent biochemical and immunohistochemical studies have shown that the opioid peptides, enkephalins, occur in nerve terminals and cell bodies in mammalian sympathetic ganglia1-3. Opiates and enkephalins are thought to inhibit synaptic transmission in the peripheral nervous tissues as well as in the central nervous system4-12. The mechanisms of the opiate actions, however, are not entirely clear; both pre- and postsynaptic sites of action have been proposed7-9,11,12. As acetylcholine is known to be the major neurotransmitter in the autonomic ganglia and as the mechanism of synaptic transmission is well clarified13, analysis of the peptide action could be more easily but equally usefully carried out in the peripheral synapses than in central synapses. We now report that enkephalins presynaptically inhibit cholinergic transmission in sympathetic ganglia.


The influence of 8-Br 3', 5'-cyclic nucleotide analogs and of inhibitors of 3', 5'-cyclic nucleotide phosphodiesterase, on noradrenaline secretion and neuromuscular transmission in guinea-pig vas deferens. The effects of 3', 5'-cyclic nucleotide phosphodiesterase (PDE) inhibitors and of 8-Br 3', 5'-cyclic nucleotide analogs on nerve-muscle transmission were studied in the guinea-pig vas deferens preincubated with 3H-noradrenaline. 8-Br cyclic AMP and the PDE inhibitors 3-isobutyl-1-methylxanthine (IBMX) and 3-propionyl-4-hydrazinopyrazolopyridine (SQ 20006) enhanced the secretion of 3H-NA evoked by transmural nerve stimulation. 8-Br cyclic GMP was without effect in this respect. The muscle contraction evoked by transmural nerve stimulation, high potassium or by application of exogenous noradrenaline was depressed by IBMX and SQ 2006. The contraction evoked by transmural nerve stimulation was enhanced by 8-Br cyclic AMP and depressed by 8-Br cyclic GMP. These findings suggest differential involvement of 3', 5'-adenosine- and guanosine-cyclic nucleotides in excitation-secretion-coupling in the noradrenergic sympathetic nerves, and in excitation-contraction-coupling in the smooth muscle, of guinea-pig vas deferens.


Effects of sympathectomy on the in vivo alpha and beta-responses of the parotid gland. Salivary secretion in response to noradrenaline and isoprenaline was measured in innervated and chronically sympathectomized parotid glands of the rat. In innervated glands, the responses to isoprenaline lasted longer than those to noradrenaline. Chronic sympathetic denervation enhanced the responses to both noradrenaline and isoprenaline. The magnitude of the supersensitivity to isoprenaline was related to the dose and the time at which the responses were analyzed. Supersensitivity was greater for the initial than for the total secretion elicited by isoprenaline. Propranolol (1 mg/kg) and phentolamine (5 mg/kg) were used in order to determine the relative participation of alpha and beta-adrenoceptors in the enhanced responses to isoprenaline. The results suggest that postjunctional supersensitivity for the secretory responses of this organ to isoprenaline is mainly mediated through beta-adrenoceptors of the secretory cells and alpha-adrenoceptors of the myoepithelial cells.


Effects of sympathectomy on the in vitro alpha and beta-responses of the parotid gland. Amylase and K+ resease were measured in slices obtained from innervated and sympathetically denervated rat parotid glands. Amylase release induced by noradrenaline was found to be increased after chronic denervation. The postjunctional component of supersensitivity to noradrenaline was evidenced by an increase in the maximal response to this agonist. The maximal response of denervated glands was blocked by propranolol 10(-5) M. On the other hand, chronic denervation did not modify the alpha-adrenoceptor-mediated response, K+ release, either in the presence or in the absence of ouabain. It is concluded that, after sympathetic denervation, postjunctional supersensitivity develops for the beta-adrenoceptor-mediated but not for the alpha-adrenoceptors-mediated effects of noradrenaline.


Indirect evidence for presynaptic location of opiate receptors on chemosensitive primary sensory neurones. 1. Rats were pretreated with 50 mg/kg s.c. capsaicin or solvent on the second day of life; 5 months later 3H-diprenorphine binding to homogenates of the whole spinal cord or of the upper dorsal horn of the spinal cord was investigated. 2. Capsaicin pretreatment resulted in a 17% decrease of opiate binding sites in the whole spinal cord and a 37% decrease in the upper dorsal horn with no change in their affinity. 3. Since neonatal capsaicin pretreatment causes degeneration of chemosensitive primary sensory neurones it is concluded that some opiate receptors are located presynaptically on the central terminals of these neurones.


Intraneural ganglion of the common peroneal nerve. The authors report a fifteen-year-old patient with an intraneural ganglion cyst of the common peroneal nerve, with paralysis of the anterior tibial muscles which was subacute in onset. The lesion was radically removed using microtechniques. Such a purely intraneural location has rarely been described. The use of the surgical microscope and pathological study of the peroneal articular branches allows one to reject the possible synovial origin of this lesion. The different hypotheses regarding aetiology are discussed, as well as the possible lines of treatment.


Classification of tumours in the sellar region using CT and encephalotomography. Tumours in the sellar region may still present diagnostic problems. We therefore compared encephalotomographic and CT findings in sellar tumours and 91 patients were examined by both methods. For clinical purposes and operative treatment the degree of compression of the hypothalamus is of great importance. Encephalotomographic and CT findings are analysed and demonstrated. In cases of density differences between tumour and brain tissue shown by contrast enhancement, more information on the inner structure of a tumour (cystic or solid) can be obtained by computerized tomography. But if these are no density differences or/and contrast enhancement, tumour diagnosis is made easier by encephalotomography because of the great efficiency of this method.


Radiotherapy of intracranial astrocytomas: analysis of 417 cases treated from 1960 through 1969. In a review of 417 intracranial astrocytomas treated radiotherapeutically at the Mayo Clinic from 1960 through 1969, the well-known correlation of tumor grade with survival was verified. Totally unexpected was the finding that age was fully as important a discriminant as tumor grade. Another unexpected finding was that patients treated with biopsy only followed by radiation therapy did as well as or slightly better than those subjected to resection followed by postoperative radiotherapy. We could not verify the importance to survival of either large dose or large volume radiotherapy, which has been emphasized by some. Patients receiving less than 1400 rets did just as well as or slightly better than those receiving more than 1400 rets. With low grade astrocytomas, survival beyond 4 years was significantly worse (higher death rates) in the group receiving more than 1400 rets. This suggested the possibility of radiation damage with delayed manifestations. We also could not verify an increased effectiveness for the generally accepted use of total brain irradiation for high grade gliomas.


Simian virus 40-related DNA sequences in a human brain tumor. Papovaviruses can induce experimental brain neoplasms in animals, and some papovaviruses have been implicated in the formation of various human tumors. We examined a series of seven human brain tumors removed at craniotomy for the presence of viral DNA sequences by the technique of DNA-DNA hybridization. Simian virus 40 (SV40) DNA was labeled in vitro and used as a "probe" for detecting related DNA sequences in cellular DNA extracted from brain tumors. SV40-related DNA sequences were found in DNA extracted from one tumor, a glioblastoma multiforme. It was calculated that approximately 1.2 viral genome equivalents per diploid cell were present in the tumor. Since the rate of reassociation of the probe deviated from ideal second-order kinetics, it is surmised that either only a portion of the SV40 genome was present in tumor cells or, alternatively, that the probe detected a related human papovavirus.


Electron microscopy of skin and peripheral blood lymphocytes in infantile (Santavuori) neuronal ceroid lipofuscinosis. Skin punch biopsies and peripheral blood lymphocyte preparations from two siblings with early infantile (Santavuori) neuronal ceroid lipofuscinosis have been examined by electron microscopy. In both cases characteristic osmiophilic inclusion bodies were found in various cells in the skin and in lymphocytes. In one case, lymphocyte inclusions were detected before the onset of any symptoms as a result of a family study. This indicates the possibility of screening lymphocytes of siblings of affected patients as a result of a family study. This indicates the possibility of screening lymphocytes of siblings of affected patients with a view to early detection of the disorder.


[Trophoblastic disease in one ovum in a twin pregnancy]. A case of trophoblastic disease in a twin pregnancy is described from its diagnosis to follow-up after emptying of the uterine cavity. The possible causes of trophoblastic disease are discussed.


False-positive amniotic fluid cytology in a parturient with active genital herpes infection at term. A patient who developed genital herpes infection diagnosed at 37 weeks' gestation is presented. Amniocentesis performed at 38 weeks' gestation revealed herpetic changes in fetal cells. Subsequent amniotic fluid viral culture was negative. The infant was delivered by elective primary cesarean section at 39 weeks' gestation, demonstrated no evidence of herpetic infection, and was well at a 3-month follow-up examination. Positive cytologic changes seen in epithelial cells obtained by amniocentesis do not necessarily indicate intrauterine infection of the fetus. Cytologic changes alone are inadequate, and viral isolation by culture or by immunofluorescence should be required before prophylactic cesarean section is abandoned in the treatment of the parturient with active genital herpes infection near term.


Herpesvirus hominis type 1: a summary of structure, composition, growth cycle, and cytopathogenic effects. The widespread occurrence of localized herpetic infections constitutes a significant problem for the dental profession. A summary of the structure and composition, growth cycle, and cytopathogenic effects of Herpesvirus hominis Type 1 is offered with the hope that a greater understanding of herpetic infections will ensue.


Fractures of the malar bone. Follow-up study based upon a questionnaire. During the years 1969-1976 214 patients with malar bone fractures were treated. A retrospective study was carried out and a follow-up by the aid of a questionnaire. The clinical judgement at 1 week after surgery showed good results. The follow-up study revealed, however, a poorer picture. Among the surgically treated patients who answered the questionnaire, 89 of 143 reported different complaints. Anaesthesia in the check and diplopia were the most common complaints. It seems anyhow that the process of regeneration of the sensible nerve takes longer than predicted. The pre-operative evaluation of the eye motility is of utmost importance.


[Relations of HLA A2, A3, B12 antigens, and A2-B12 supposed haplotype association, with latent herpes virus infection in patients with chronic renal disease (author's transl)]. Comparison of 105 patients suffering of chronical renal failure with 108 normal persons showed that, in the former group, a relation could be established between HLA A2, A3, and B12 histocompatibility antigens as well as haplotype association A2-B12, and chronic infection by herpes virus. The high number of herpes virus observed in patient group bearing the BW 35 antigen was discussed.


Malignant fibrous histiocytoma of the larynx. Fibrous histiocytoma is a rare mesenchymal tumor. The origin is believed to be a pleuri-potential cell, histiocytic in type, which can evolve into a facultative fibroblast under certain conditions. A case of a malignant fibrous histiocytoma in the subglottic larynx is reported. At present, the clinical behavior and degree of malignancy cannot be predicted. It appears that wide, local excision of the tumor with a margin of normal tissue is the treatment of choice. Regional block dissection of lymph nodes is not indicated unless clinically palpable nodes are present.


Sarcomatous degeneration in a nasopharyngeal angiofibroma. Malignant degeneration in a juvenile nasopharyngeal angiofibroma has been reported in the literature in only four patients. All of these persons had been previously treated for cure with gamma irradiation. The case report of a 47-year-old man with a 31-year history of nasal obstruction is presented. A recurrence excised 18 months after initial removal of an angiofibroma revealed the surprising diagnosis of fibrosarcoma.


Recurrent adenoid cystic carcinoma of the trachea: intraluminal radiotherapy. Malignant neoplasms of the trachea are rare lesions, of which adenoid cystic carcinoma constitutes 30%. A case of extensive tumor recurrent after primary radiotherapy is presented. Surgical resection and radioisotope implantation were impossible. A method was devised for placement of intraluminal iridium 192 by casting six small hollow plastic tubes to the outer circumference of a Silastic endotracheal tube. This was an easy, safe, and effective method of delivering radiation to the trachea while sparing other vital structures and organs. Excellent tumor response and palliation were achieved. The literature is reviewed and the problems in diagnosis of this tumor are discussed. A comprehensive plan for early diagnosis is presented. All current methods of treatment are discussed, and the need for aggressive surgical management is stressed.


Lipid and lipoprotein tracking in children. Prospective studies in adults reveal that CHD risk is continuously related to total and LDL cholesterol levels (positively) and to HDL cholesterol levels (inversely) throughout their entire distribution. Such findings raise the possibility that children who tend to track in the lowest and highest decile for total and LDL cholesterol may mature to become adults respectively at reduced and increased CHD risk. Children who tend to track in the lowest and highest deciles for HDL cholesterol may also mature to become adults respectively at increased and reduced CHD risk. Although the degree of tracking at either extreme of the distribution, and for the group as a whole, appears to have adquate cohesiveness to be useful for prediction of future lipid and lipoprotein levels, individual children at the extremes of the distributions will need to be followed over longer periods of time, to identify the continuity of childhood and adult values, and eventual CHD risk.


Plasma glucocorticoid and adrenocorticotropin concentrations measured serially in growth-retarded fetal lambs. We studied the fetal pituitrary-adrenal axis in 9 growth retarded fetal lambs by serially measuring plasma glucocorticoids and ACTH concentrations from 115 to 140 days of gestation and adrenal blood flow at 138 days of gestation. At each gestational age period studied, plasma glucocorticoid and ACTH concentrations were similar in both growth retarded and control fetuses (Figs. 1, 2). However, ACTH concentration tended to be higher in the last 20 days of gestation in the growth retarded fetuses. This resulted in a significantly lower glucocorticoid/ACTH ratio between 121 to 130 days of gestation in the growth retarded fetal lambs as compared to the control fetuses (212 +/- 111 vs. 1042 +/- 257). The glucocorticoid/ACTH ratio in the growth retarded fetuses became similar to the ratio of the control fetuses at 131 to 140 days (445 +/- 145 vs. 554 +/- 182). Adrenal blood flow was significantly increased at term in the the growth retarded fetuses (535 +/- 87 vs. 302 +/- 40 ml/min/100g). These data indicate that the pattern of maturation of the fetal adrenal gland in the growth retarded fetal lamb is similar to that of the normal sized fetus. Because of the increased adrenal blood flow observed at 138 days of gestation, there is a suggestion of a decreased sensitivity to ACTH in these animals.


Effect of sleep state on chest distortion and on the ventilatory response to CO2 in neonates. In 10 preterm and 10 term infants, the effect of sleep state on chest distortion and on the ventilatory response to CO2 was assessed. It was found that chest distortion and ventilatory response to CO2 were independent of sleep state. Chest distortion, however, was more frequent in preterm than in term infants. The authors suggest that the increased prevalence of chest distortion in preterm infants is related to their highly compliant chest wall rather than to differences in sleep state.


Bactericidal and metabolic function of polymorphonuclear leukocytes. The bactericidal and metabolic function of the phagocytic system requires integration of several complex humoral and cellular factors responding to different regulators. Polymorphonuclear leukocytes are highly mobile cells, capable of phagocytosis of bacteria or fungi with formation of a "cellular digestive system" containing reactive oxygen radicals, hydrogen ions, and digestive enzymes. The unique metabolism of oxygen in neutrophils results in release of energy as light (chemiluminescence) a response closely associated with microbiol killing. Neonatal neutrophils cope with normal bacterial challenges in vitro as efficiently as adult neutrophils; however, these cells have decreased capacity for locomotion, decreased deformability, decreased phagocytosis in low serum concentrations, and decreased chemiluminescence. These subtle defects in function can be amplified by exaggerated challenge which may be related to a higher incidence of sepsis during the neonatal period.


Defective oxidative metabolism in newborn neutrophils: discrepancy between superoxide anion and hydroxyl radical generation. Investigation of oxidative metabolism in neutrophils (PMNs) from newborns was performed by measuring generation of superoxide anion (.O2-) and production of hydroxyl radical (.OH) in the resting state and after stimulation with opsonized zymosan or phorbol myristate acetate (PMA). Neutrophils from cord blood of ten term infants and normal adult controls were tested simultaneously. Cord PMNs generated significantly more .O2- than paired adult controls when stimulated with opsonized zymosan (P less than .01) and produced less .OH with PMA (P less than .005). When the amount of .O2- and .OH released by newborn PMNs with both stimuli was expressed as percent of values obtained from paired adult controls, there was a discrepancy in the generation of these two radicals: newborn PMNs produced relatively less .OH compared to .O2-. This decreased ability to produce .OH could underlie defective bactericidal activity in PMNs of neonates.


Reciprocal responses to tilt of neurones within both fore- and hindlimb regions of Deiters' nucleus. In decerebrate cats with cerebellum intact the frequency response of 102 neurons located within the lateral vestibular nucleus (LVN) to sinusoidal stimulation of vestibular receptors was analyzed. Positional sensitive units, showing a reciprocal pattern of response to lateral tilting, characterized by an excitation during ipsilateral and a depression during contralateral tilt, were equally found in the rostroventral (forelimb) and dorsocaudal (hindlimb) divisions of the LVN. No unit was found to be excited during both ipsilateral and contralateral tilts. A comparison between these findings and those reported in cerebellectomized preparations indicates that the reciprocal pattern of response to tilt of neurons, particularly located in the hindlimb region of the LVN, depends upon the anatomical integrity of the cerebellum.


Effect of repetitive stimulation on the frog neuromuscular transmission. Presynaptic and postsynaptic effects on the neuromuscular transmission were studied during 20 min of indirect stimulation at 10/s. During the 'facilitation' period, there was an increase in the quantal content, in the frequency of miniature endplate potentials and in their amplitude. All these parameters were decreased during the 'depression' period. Besides, the end-plate current (e.p.c.), recorded during this high rate of stimulation, increasingly lengthened. The falling phase of the e.p.c. was exponential during facilitation, while marked deviations from the exponential time course were observed during depression. The experiments showed that a possible change in the kinetics between the receptors and the mediator was not responsible for the lengthened time course of the e.p.c. Therefore, it is assumed that either the delayed diffusion of the transmitter from the synaptic cleft or an altered mechanism of the release of acetylcholine may be involved. The latter possibility is supported by a progressive prolongation of the synaptic delay, which was observed during a prolonged repetitive stimulation.


[Immunological abnormalities in atopic dermatitis (author's transl)]. The humoral immunological abnormalities in atopic dermatitis (AD) are mainly represented by the increase of IgE. But high IgE levels are inconstant and non specific for AD. They may remain elevated during remission and their role in the pathogenesis is unclear. The origin of the stimulation of IgE-producing B lymphocytes could be a transcient deficiency of IgA in newborns or a defect of the T cell control. A defect of cell-mediated immunity has been clearly demonstrated by numerous in vitro studies indicating low percentages of E rosettes and low stimulation by mitogens. This defect could be a T suppressor cell defect. The correlations of these immunological abnormalities with the Sczentivanyi's theory as well as the role of immuno-pharmacological disturbances in the clinical picture and the possible new treatments of AD are discussed.


Comparative study of papovavirus DNA: BKV(MM), BKV(WT) and SV40. Extensive physical mapping revealed that approximately 90% of the genomes of BKV(prototype, WT) and BKV (MM strain) are identical or closely related. Nucleotide sequences of the non-homologous regions and a large portion of the homologous regions have been determined for both genomes. The coding sequence of small t antigen of BKV(MM) is 216 nucleotides shorter than that of BKV(WT), even though no differences in biological function of the t antigen was observed. Both genomes contain three similar sets of 44-61 base-pair repeated sequences. However, the DNA sequence of the tandem repeats is totally different between BKV (human cell as host) and SV40 (monkey cell as host). On the other hand, the region between the N-terminus of the T antigen genes and the origin of replication is dominated by a similar set of palindromic sequences in BKV and SV40 DNA. There is also extensive homology between the regions which code for proteins in BKV and SV40, suggesting a close evolutionary relationship.


Nicking-closing enzyme is associated with SV40 DNA in vivo as a sodium dodecyl sulfate-resistant complex. A fraction of the cellular nicking-closing (NC) enzyme cosediments with SV40 chromatin isolated after Triton X-100 treatment of infected cells nuclei. Extraction of viral DNA according to the Hirt procedure by treatment of infected cells with sodium dodecyl sulfate (SDS) followed by sedimentation in sucrose gradient to separate the DNA from the bulk of detergent also revealed NC activity associated with DNA. Reconstitution experiments showed that only prebinding of the NC enzyme to DNA protects it against irreversible inactivation by SDS. These results suggest that a fraction of the cellular NC activity is indeed associated with the viral chromosome in vivo.


Preferential in vitro assembly of nucleosome cores on some AT-rich regions of SV40 DNA. We have found that nucleosomes reconstituted from histone octamers and SV40 DNA Form I by progressively decreasing the salt concentration from 2 M NaCl are formed preferentially around 0.27, 0.37, 0.50 and 0.85 on SV40 DNA (relative to the EcoRI site). When SV40 DNA Form III is used, the nucleosomes form mainly at 0.28, 0.38, 0.61 and 0.83. These sites are very close to both the sites of RNA chain initiation by calf thymus RNA polymerase B on SV40 DNA Form I (0.25, 0.35, 0.42 and 0.88) and the regions of the supercoiled DNA which are readily denaturable by T4 gene 32 protein (0.25, 0.47 and 0.88), and correspond to AT-rich regions as deduced from the nucleotide sequence of SV40 DNA. The physiologically important region around 0.67 is an unfavourable site for all three types of proteins, and corresponds to a GC-rich region surrounding a 17 base pair AT cluster.


Polyoma virus. The early region and its T-antigens. The DNA sequence of the early coding region of polyoma virus is presented. It consists of 2739 nucleotides. The sequence predicts that more than one reading frame can be used to code for the three known polyoma virus early proteins (designated small, middle and large T-antigens). From the DNA sequence, the 'splicing' signals used in the processing of viral RNA to functional messenger RNAs can be predicted, as well as the sizes and sequences of the three proteins. Other unusual aspects of the DNA sequence are noted. Comparisons are made between the DNA sequences and the predicted amino acid sequences of the respective large T-antigens of polyoma virus and the related virus Simian Virus (SV) 40.


Hybrid plasmids containing an active thymidine kinase gene of Herpes simplex virus 1. The gene for the thymidine kinase (TK) of Herpes simplex virus type 1 (HSV-1) is located in the KpnI m and BamHI p fragments of the genome (Wigler et al., Cell 11, 223-232 (1977)). These fragments have been inserted into the EcoRI and BamHI sites, respectively, of plasmid pBR322, and propagated in E.coli. The TK gene contained in the recombinant plasmids was shown to be biologically active when introduced into TK- mouse L cells. Detailed restriction site maps of the BamHI p fragment have been constructed and the approximate location of the TK gene has been determined. Mouse cells transformed with cloned HSV-1 tk+ DNA produced HSV-1-specific thymidine kinase; superinfection with HSV-1 tk- virus increased the level of TK activity tenfold, suggesting that the BamHI p sequences present in transformed cells respond to virus-encoded regulatory gene product(s).


Specific chemical labeling of DNA fragments. We describe a simple method for specific chemical labeling of DNA fragments at their 3'-termini. The procedure includes enzymatic addition of 4-thiouridine, followed by reaction in mild non-denaturing conditions with the highly reactive alpha-haloacetamido derivatives of several chemical labels. The attached reporter molecule can be removed by extended treatment with beta-mercaptoethanol. Among the potential applications of this labeling method is the study of specific protein-DNA interactions in solution.


Replicative activity of histone-deficient SV40 chromatin. Histone-deficient SV40 chromatin, selectively radiolabeled in the DNA following the addition of cycloheximide to infected monkey cells, was compared with the normal 55S viral chromatin for its ability to serve as a template for a subsequent round of replication. After the removal of cycloheximide, the 26S histone-deficient SV40 chromatin was converted to apparently normal 55S chromatin. During this conversion, the chromatin which sedimented at 26-40S failed to replicate whereas the 44-55S chromatin contained a large fraction (28%) of newly replicated DNA molecules. Thus, the DNA in the 26S histone-deficient 40S chromatin cannot replicate without the prior and/or concommitant addition of protein which increases its sedimentation rate to 41-55S. Nevertheless, when compared with normal 55S viral chromatin, the histone-deficient SV40 chromatin had nearly a 3-fold greater probability of functioning as a template for a subsequent round of replication.


Non-specific interactions of CRP from E. coli with native and denatured DNAs: control of binding by cAMP and cGMP and by cation concentration. The cyclic adenosine 3',5'-monophosphate receptor protein of Escherichia coli (CRP) binds cooperatively to single- and double-stranded DNA. Binding data could be fitted to the model of McGhee and von Hippel (1) and show that neither strandedness of DNA, nor the effectors cAMP and cGMP or the ionic strength (KCl) do change appreciably the cooperativity parameter omega (omega approximately or equal to 100), and site size of DNA. Instead, distinctly different slopes were observed for the linear decrease of log K omega (a measure of the overall affinity) as a function of log (K+). From these double-log plots (2), the number of cations released and the non-electrostatic contributions to the binding free energy could be determined. Binding of CRP to single-stranded DNA is slightly favored under physiological ionic conditions (0.15-0.20 M), but such a preferential binding is almost abolished in the presence of cAMP which increases the strength of the interaction of the protein with both forms of DNA. CGMP does not change the binding properties and interactions of CRP with DNA. These observations do not support the proposal that the cAMP-CRP complex could stimulate transcription via some "melting" property unless its interactions be dramatically changed when it binds specifically to promoter DNA.


The extraction from the blood of phosphate compounds by canine bone. Uptake by bone of 99mTc-labeled pyrophosphate (PPi) and ethane-1-hydroxy-1,1-diphosphonate (EHDP) involves passage through capillaries in the Haversian system, extracellular fluid space, and membrane of osteoblasts before adsorption onto available apatite surfaces. Passage through capillaries was studied by the outflow-dilution technique in a canine tibia model in mongrel dogs. 51Cr-labeled albumin (nondiffusible reference tracer), sucrose (diffusible reference tracer), PPi, and EHDP were injected into the isolated nutrient tibial artery. Blood was collected from the ipsilateral femoral vein every 15 sec for 2 min. Emax, the apparent extraction at the time of the peaks of the dilution curves, is influenced little by back diffusion; for PP, Emax (mean +/- SD) = 0.42 +/- 0.08 (N = 4); for EHDP, Emax = 0.27 +/- 0.05 (N = 10). Net extraction (apparent fractional retention by bone at 2 min) was 0.36 +/- 0.1 for PP. Emax for 85Sr chloride and 18F sodium in our laboratory is 0.69 +/- 0.11 (N = 14) and 0.70 +/- 0.008 (N = 9), respectively. Permeabilities (P) were calculated by PS = -Fs loge (1-Emax), where Fs = plasma flow and S = capillary surface; the ratio of P for EHDP to P for sucrose was 0.71 which is similar to the ratio of diffusion coefficients, 0.78. The data suggest that EHDP and probably also PPi pass through the capillaries by passive diffusion. As expected, the extraction through capillary walls for EHDP and PPi was lower than that for 85Sr and 18F because of their larger molecular size.


[Systematic evaluation of 8 arginine vasopressin and neurophysins plasma levels in 10 patients with oat cells carcinoma of the lung (author's transl)]. Plasma AVP level, neurophysins and osmolality have been measured in 10 patients with oat cell carcinoma of the lung but without any biological signs of syndrome of inappropriate secretion of ADH (SIADH), before and 15 mn after an intravenous injection of Ethanol. No statistically significant difference was noted in the AVP, neurophysins and osmolality values between 10 patients with asymptomatic oat cell carcinoma and control population (10 normal volunteers, 12 patients with non neoplasic lung pathology and 10 patients with different lung carcinoma). We concluded that AVP and neurophysins cannot be considered as a good tumoral marker in the detection of oat cell carcinoma of the lung.


[Endobronchial Abrikosov's tumor. Two new cases associated to malignant tumors]. Endobronchial localizations of the granular cell tumours or Abrikossoff's tumour are very rare (6%) but their association with malignant tumours is exceptional and perhaps fortuitous. The clinical manifestations are generally the consequence of bronchial erosion and blocking. Bronchial endoscopy is the essential examination which allows the tumour to be seen and a biopsy to be made. Pathological examination confirms the diagnosis. The typicall cell of granular cell tumour is a large, polygonal one with finely granular eosinophilic cytoplasm and a small dark vesicular nucleus. Such cells are arranged in syncytial masses containing long, spindleshape cells with elongated nuclei. If the optical and elctron microscope description is well known, the histogenesis of the disease is still in dispute; the neurogenic origin seems to be the best one, but at present there is not enough evidence to make this assumption a certainty. Surgical exeresis is the only therapy to bring about a cure, but clinical supervision is necessary because of a possible relapse.


An interferon-induced phosphodiesterase degrading (2'-5') oligoisoadenylate and the C-C-A terminus of tRNA. A phosphodiesterase characterized by a generally higher activity on 2'-5' than on 3'-5' phosphodiester bonds was isolated from mouse L cells treated with interferon. A similar enzyme was purified from mouse reticulocytes. The phosphodiesterase 2'-PDi splits the 2'-phosphate bond of pppA2'p5'A2'p5'A, the oligonucleotide activator of ribonuclease F. The level of phosphodiesterase 2'-PDi is increased by interferon treatment of L cells. The phosphodiesterase was also shown to degrade the C-C-A terminus of tRNA and to reduce the amino acid acceptance of tRNA in cell-free extracts, thereby causing a tRNA-reversible inhibition of mRNA translation.


Sequences of vesicular stomatitis virus RNA in the region coding for leader RNA, N protein mRNA, and their junction. The RNAs extracted from purified preparations of the Indiana and New Jersey serotypes of vesicular stomatitis virus were polyadenylylated in vitro by using polynucleotide phosphorylase and sequence determination was carried out by the dideoxynucleotide method using reverse transcriptase and dT8AC primer. On both virus RNAs a short stretch of adenylic acid residues is present between the regions coding for the leader and N protein mRNAs. Other features of the RNA sequences of the two viruses are compared to each other and to published data.


Integration of bovine leukemia virus DNA in the bovine genome. DNA preparations from circulating leukocytes, lymph node tumors, and spleens of three bovine leukemia virus-infected cattle were fractionated by Cs2SO4/3,6-bis(acetatomercurimethyl)dioxane density gradient centrifugation. Bovine leukemia virus proviral sequences were found in large GC-rich fragments having a buoyant density in CsCl close to 1.708 g/cm3. Provirus integration, therefore, does not take place at random locations in the host genome, but in a specific class of DNA segments. Hybridization of cDNA synthesized on viral RNA to EcoRI and Xba I restriction fragments of the DNA from infected cells showed that: (i) only one copy of proviral DNA is integrated per haploid genome; (ii) different restriction patterns were found in the proviral DNAs present in the genomes of different animals, providing evidence for the existence of several strains or mutants; and (iii) different integration sites for the proviral DNA were found in the genome of different animals and of different infected cells in the same animal. The latter finding strongly suggests a polyclonal origin of bovine leukemia virus-infected cells.


Escherichia coli heat-labile enterotoxin: DNA-directed in vitro synthesis and structure. Escherichia coli heat-labile enterotoxin was synthesized in a cell-free system directed by DNA of the plasmid P307. Synthesis of the toxin, assayed by the elongation induced in Chinese hamster ovary cells, was strongly stimulated by cyclic AMP and occurred at physiological levels of Mg2+ only when the polyamine spermidine was present. Activity was abolished by heat and antisera prepared against the enterotoxins of both E. coli P263 and Vibrio cholera. Tritium-labeled enterotoxin was purified by immunoprecipitation and examined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. When gel slices were assayed for the ability to stimulate adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] activity in erythrocyte ghosts, two peaks were found, one at Mr 26,000 and frequently, but not always, another at Mr 23,000. Detection of radiolabeled protein by fluorography and scintillation counting of gel slices revealed three prominent polypeptides, two corresponding to the peaks having adenylate cyclase-stimulating activity and a further one of Mr 11,500, identical to that of the cholera subunit B. The data suggest that the E. coli heat-labile enterotoxin synthesized in the cell-free system has a subunit structure.


Origin of observed changes in 14N hyperfine interaction accompanying R leads to T transition in nitrosylhemoglobin. Theoretical investigations of electronic distributions in eight different structural forms of nitrosylhemoglobin were carried out to study the changes in (14)N hyperfine interaction observed with the transition from R to T structures under the influence of inositol hexaphosphate or changing pH. Four of the eight forms studied consisted of protonated and deprotonated N(pros) in the proximal imidazole ligand with linear and bent Fe-N-O structures. Two other forms had a straight Fe-N-O structure and Fe-Im bond stretched by 0.5 and 1.0 A. The other two systems we have studied are five-liganded NO-heme with bent and straight Fe-N-O structures. Our investigations show that arrangements of energy levels did not differ significantly among all the structures, the unpaired electron always occupying an antibonding orbital with d(z)2 symmetry. The protonated and deprotonated systems with either linear or bent Fe-N-O structure showed substantial hyperfine interaction of the (14)N nuclei of the NO group and the N(epsilon) atom of the proximal imidazole, indicating that a 9-line electron spin resonance hyperfine pattern (R structure) would be expected in all four cases. On the other hand, the extensions of the Fe-Im bond produce a sizeable decrease in the (14)N(epsilon) hyperfine interaction, indicating that an extension beyond 1.0 A would provide a 3-line hyperfine pattern close to that found for the five-liganded NO-heme system. Our results thus provide quantitative support for the model of severe extension or cleavage of the Fe-N(epsilon) bond proposed in the literature for explaining the R-to-T transition of the alpha-chain of nitrosylhemoglobin.


Rate limitation of (Na+ + K+)-stimulated adenosinetriphosphatase by membrane acyl chain ordering. A somatic cell mutant (CR1) of the Chinese hamster ovary cell line (CHO-K1) that is defective in the regulation of cholesterol biosynthesis can be grown under conditions in which plasma membranes from these cells display various cholesterol contents and acyl chain order parameters. The (Na+ + K+)-stimulated adenosinetriphosphatase (ATP phosphohydrolase, EC 3.6.1.3) from these cells was shown to vary in activity by a factor of 10 as the order parameter was varied, and the activity exhibited an exponential dependence on this parameter. Under these conditions the number of Na+,K+-ATPase molecules was shown to remain constant by affinity labeling with [gamma-32P]ATP in the absence of Na+. Control experiments showed that alteration in cholesterol content without change in order parameter did not result in altered enzyme activity. It is concluded that, under our conditions, the rate of catalysis by the Na+,K+-ATPase is determined by the order parameter. These studies suggest a physical mechanism by which variation of membrane lipid composition or other factors that determine membrane lipid acyl chain order parameter can result in variation in membrane enzyme activity.


Sequential adsorption-electrophoresis: combined procedure for purification of calcium-dependent cyclic nucleotide phosphodiesterase. A procedure for combined sequential affinity adsorption-electrophoresis has been devised. Its use for the rapid purification of a calcium-dependent cyclic nucleotide phosphodiesterase from bovine brain in high yield is described. In this procedure, proteins bound to a solid phase of calcium-dependent regulatory protein (CDR) linked to Sepharose 4B were electrophoretically eluted, concentrated, and separated, thus avoiding the large losses in activity incurred during attempts to purify further the phosphodiesterase eluted by conventional means. The highly purified phosphodiesterase prepared by this method was stable for months at -60 degrees C in the presence of glycerol. It has a higher affinity for cyclic GMP than for cyclic AMP, and hydrolysis of both substrates is stimulated 5- to 6-fold by calcium plus CDR. Factors that influence adsorption of the enzyme to CDR-Sepharose and selection of optimal conditions for electrophoresis were investigated. Sequential adsorption-electrophoresis should be generally useful in the purification of macromolecules for which affinity adsorbents are available. The procedures described here could be directly applicable to the purification of proteins that, like the phosphodiesterase, interact with CDR.


Regulation of activity of chromatin receptors for thyroid hormone: possible involvement of histone-like proteins. Thyroid hormone receptors lose their capability for high-affinity binding of the biologically active triiodothyronine after solubilization and separation from other chromatin proteins. The high-affinity triiodothyronine-binding capacity can be reconstituted by addition of a histone-containing extract of chromatin of purified core histones (H2A, H2B, H3, and H4); a number of other acidic or basic proteins tested were ineffective. The data support a model of the receptor in which a "core" receptor subunit that contains a thyroid hormone-binding site interacts with a regulatory subunit, which is possibly a histone or histone-like species. This interaction with the "core" subunit enables the resulting "holo" receptor to bind biologically active hormones. These data also suggest that histones or related proteins can modulate the activity of nonhistone chromosomal proteins that are involved in regulating the expression of specific genes.


Squalene synthetase activity in human fibroblasts: regulation via the low density lipoprotein receptor. Squalene synthetase (farnesyltransferase; farnesyl diphosphate:farnesyl-diphosphate farnesyltransferase, EC 2.5.1.21), the enzyme in the cholesterol biosynthetic pathway that converts farnesyl pyrophosphate into squalene, is subject to regulation in cultured human fibroblasts. When cholesterol-carrying low density lipoprotein (LDL) was removed from the serum of the culture medium, squalene synthetase activity increased 8-fold over 24 hr. When LDL was added back to the medium, squalene synthetase was slowly suppressed, 50% and 90% reduction occurring in 15 and 48 hr, respectively. Suppression of squalene synthetase required uptake of LDL via the LDL receptor; hence, it did not occur in mutant fibroblasts from a patient with homozygous familial hypercholesterolemia that lack receptors. The addition of a mixture of 25-hydroxycholesterol and cholesterol suppressed squalene synthetase equally well in normal and mutant fibroblasts. Coupled with previous data, the current findings indicate that cholesterol derived from LDL regulates at least two enzymes in the cholesterol synthetic pathway in fibroblasts: (i) its primary action is to rapidly suppress 3-hydroxy-3-methylglutaryl coenzyme A reductase [mevalonate:NADP(+), oxidoreductase (CoA-acylating), EC 1.1.1.34], which reduces mevalonate production by 95% within 8 hr, and (ii) its secondary action is to slowly suppress squalene synthetase. The LDL-mediated suppression of squalene synthetase does not regulate de novo cholesterol synthesis; it occurs after 3-hydroxy-3-methylglutaryl coenzyme A reductase is already suppressed. Rather, we hypothesize that it may function to allow the pool size of farnesyl pyrophosphate to be maintained in the presence of LDL so that low levels of mevalonate can be shunted preferentially into nonsterol products, such as ubiquinone-10 and dolichol. This mechanism may explain the earlier observation that the synthesis of ubiquinone-10 in fibroblasts proceeds at a normal rate in the presence of LDL despite a 95% decrease in mevalonate production.


Structure and specific sequences of avian erythroblastosis virus RNA: evidence for multiple classes of transforming genes among avian tumor viruses. Two major RNA species were found in several clonal isolates of avian erythroblastosis virus (AEV) and avian erythroblastosis-associated helper virus (AEAV) complexes: one of 8.7 kilobases (kb), the other of 5.5 kb. The 5.5-kb species was identified as AEV RNA because (i) it was absent from non-transforming AEAV isolated from the same virus complex, (ii) it was present in complexes of AEV and different helper viruses, and (iii) its structure is similar to that of avian acute leukemia viruses of the MC29 group. Molecular hybridization indicated that 54% of AEV RNA is specific and 46% is related to other viruses of the avian tumor virus group, particularly to AEAV, therefore termed group-specific. The genetic structure of AEV RNA was deduced by mapping oligonucleotides representing specific and group-specific sequences and by comparing the resulting map to maps of AEAV and of other avian tumor viruses derived previously. AEV RNA contains a gag gene-related, 5' group-specific section of 1 kb, an internal AEV-specific section of 3 kb unrelated to any other viral RNA tested, and a 3' group-specific section of 1.5 kb. The 5' section of AEV RNA is closely related to analogous 5' sections of the MC29 group viruses and is homologous with a 5' RNA section that is part of the gag gene of AEAV. The 3' section is also shared with AEAV RNA and includes a variant C-oligonucleotide near the 3' end that is different from the highly conserved counterparts of all other exogenous avian tumor viruses. By analogy with Rous sarcoma virus and the acute leukemia viruses of the MC29 group, the internal specific section of AEV RNA is thought to signal a third class of onc genes in avian tumor viruses. Comparisons with AEAV and the MC29 group viruses suggest that both the 5' gag-related and the internal specific RNA sections of AEV are necessary for onc gene function.


Characterization of the protein kinase activity of avian sarcoma virus src gene product. The avian sarcoma virus src gene product, p60src, has been purified 650-fold from cytoplasmic extracts of the rat tumor cell line RR1022 by using ammonium sulfate fractionation, hydrophobic chromatography on omega-aminohexyl agarose, and ion exchange chromatography on phosphocellulose. Partially purified p60src is a monomer, with a native molecular weight of about 60,000 and an apparent pI of 6.0. In immunoprecipitates, p60src catalyzed phosphorylation of anti-p60src IgG heavy chains within the variable (VH) domain, which contains the heavy chain portion of the antigen combining site. Crude preparations of p60src contained phosphatase activity able to cleave phosphate from IgG heavy chains; this activity was removed by the purification procedure, and partially purified p60src could phosphorylate the heavy chain of specific antibody in solution. Furthermore, purified p60src catalyzed phosphorylation in solution of the general protein kinase substrate, alpha-casein, strengthening the hypothesis that it may in fact function as a protein kinase in vivo.


A new endogenous primate type C virus isolated from the Old World monkey Colobus polykomos. A new, genetically transmitted retrovirus has been isolated from the Old World monkey Colobus polykomos. This virus, designated CPC-1, is readily transmitted to both feline and human cells in culture. Nucleic acid hybridization studies reveal that there are 50-70 copies of the CPC-1 genome in colobus cellular DNA. Related virogene sequences can be detected in the DNA of all other Old World monkeys, as well as in the DNA of at least one ape species, the chimpanzee, indicating that this virus has been genetically transmitted in primates for 30-40 million years. CPC-1 is partially related to the type C virus previously isolated from stumptail monkeys (MAC-1). These two viruses have nucleic acid sequence homology, antigenic crossreactivity in their major viral structural protein, and a very similar host range in vitro. CPC-1 and MAC-1 therefore belong to the same class of genetically transmitted primate type C viruses and, as such, represent the first example in primates of analogous endogenous retroviruses isolated from two distantly related species.


Cell-free synthesis and processing of a putative precursor for mitochondrial carbamyl phosphate synthetase I of rat liver. Total RNA or poly(A)(+) RNA of rat liver was translated in a rabbit reticulocyte or wheat germ protein-synthesizing system and the carbamyl phosphate synthetase I [carbamoyl-phosphate synthetase (ammonia); carbon dioxide: ammonia ligase (ADP-forming, carbamate-phosphorylating), EC 6.3.4.16] synthesized was isolated by indirect immunoprecipitation by using antibody purified on enzyme-bound Sepharose and Staphylococcus aureus cells. The in vitro product moved on sodium dodecyl sulfate/polyacrylamide gels as a polypeptide that was about 5000 daltons larger than the subunit of the mature enzyme (160,000 daltons). The same polypeptide was also obtained by direct immunoprecipitation or by a double-antibody precipitation method. The mature enzyme competed effectively with the in vitro product for interaction with anti-carbamyl phosphate synthetase I antibody. Digestion of the in vitro product by S. aureus protease gave a pattern of peptide fragments similar to that of the mature enzyme. A mitochondrial membrane preparation from rat liver converted the in vitro product into a polypeptide that comigrated with the mature subunit on sodium dodecyl sulfate gel electrophoresis. Similar proteolytic activity was not detected in either a cytosol or a microsomal fraction of rat liver. These results indicate that the enzyme is synthesized as a larger precursor which is converted to the mature form of enzyme by posttranslational processing.


Concomitant synthesis of beta-endorphin and alpha-melanotropin from two forms of pro-opiomelanocortin in the rat pars intermedia. In the pars intermedia of rat pituitary glands, two forms of a common precursor for corticotropin (ACTH) and beta-lipotropin with apparent molecular weights of 34,000 and 36,000 were resolved by sodium dodecyl sulfate/acrylamide gradient slab gel electrophoresis. High-performance liquid chromatographic analysis of [35S]methionine-labeled tryptic fragments of the two forms of the precursor revealed that both contained copies of ACTH-(1-8) and beta-lipotropin-(61-69) sequences. When biosynthetic studies were performed in the presence of tunicamycin, the 34,000- and 36,000-dalton forms were replaced by a peptide with an apparent molecular weight of 32,000. It was therefore concluded that the 34,000- and 36,000-dalton forms of the precursor represent two glycoprotein variants of similar polypeptides, differing in the number of asparagine-linked carbohydrate moieties. During pulse-chase incubations with [35S]methionine, the precursor forms were cleaved into two major groups of labeled products: (i) beta-endorphin and (ii) a mixture of ACTH fragments closely related to alpha-melanotropin. No ACTH-(1-39) was found at the end of a 2-hr chase period, suggesting that ACTH is not a significant hormone product of the rat pars intermedia.


Interaction site of Escherichia coli cyclic AMP receptor protein on DNA of galactose operon promoters. Cyclic AMP (cAMP) and its receptor protein (CRP) have a dual role in the regulation of the two promoters that control the galactose (gal) operon of Escherichia coli. One promoter, P1, requires cAMP-CRP for activity; the other, P2, is inhibited by these factors. We have examined the interactions site of cAMP-CRP on gal DNA by using two types of protection experiments, involving DNase digestion and methylation by dimethyl sulfate. Our results indicate that cAMP-CRP binds to gal DNA in a segment located between 50 and 24 base pairs preceding the P1 start point for transcription. Although the location of the cAMP-CRP interaction site is clearly different in gal and lac DNA, comparison of the DNA sequences suggests a similar recognition sequence. The location of the cAMP . CRP-binding site in gal further suggests that protein-protein interactions between RNA polymerase and cAMP . CRP play an important role in transcription initiation at the gal and possibly other cAMP-dependent promoters.


Beta-turns in nascent procollagen are sites of posttranslational enzymatic hydroxylation of proline. The selective hydroxylation of proline residues in nascent procollagen chains by prolyl hydroxylase (EC 1.14.11.2) can be understood in terms of the conformational feature of the -Pro-Gly-segments in linear peptides and globular proteins. The folded beta-turn conformation in such segments appears to be the conformational requirement for proline hydroxylation. The available data on the hydroxylation of native and synthetic substrates of prolyl hydroxylase are explained on the basis of the extent of beta-turn formation in them. Taken in conjunction with the conformational features of the hydroxyproline residue, our results bring out the conformational reason for the posttranslational proline hydroxylation which, it is proposed, leads to the "straightening" of the beta-turn segments into the linear triple-helical conformation.


Triplet states in photosystem I of spinach chloroplasts and subchloroplast particles. We report light-induced electron paramagnetic resonance triplet spectra from samples of chloroplasts or digitonin photosystem I particles that depend upon the dark redox state of the bound acceptors of photosystem I. If the reaction centers are prepared in the redox state P-700 A X- FdB-FdA-, then upon illumination at 11K we observe a polarized chlorophyll triplet species which we interpret as arising from radical pair recombination between P-700+ and A-. This chlorophyll triplet is apparently the analog of the PR state of photosynthetic bacteria [Parson, W.W. & Cogdell, R.J. (1975) Biochim. Biophys. Acta 416, 105-149]. If the reaction centers are prepared in the dark redox state P-700 A X FdB-FdA-, then upon illumination at 11K we observe a different triplet species of uncertain origin, possibly pheophytin or carotenoid. This species is closely associated with the photosystem I reaction center and it traps excitation when P-700 is oxidized.


Adrenocorticotropin (ACTH) induces phosphorylation of a cytoplasmic protein in intact isolated adrenocortical cells. In 32P incorporation experiments with intact adrenocortical cells, adrenocorticotropin (ACTH) or adenosine 3',5'-cyclic monophosphate (cAMP) induced a rapid and transient increase of approximately 300-500% in the phosphorylation of a 32P-containing cytoplasmic protein of about 150,000 daltons (APS150). Half-maximal stimulation of APS150 phosphorylation was observed with about 3 pM ACTH. Receptor-bound cAMP, corticosterone production, and the appearance of phosphorylated APS150 increased in parallel with respect to both time and ACTH concentration. All three responses were dependent on extracellular calcium. Inhibition of protein synthesis with cycloheximide suggested a half-life of APS150 of about 10 min. The time course of 32P incorporation into ACTH-induced APS150 in the absence and presence of nonradioactive phosphate shows that the phosphorylation of APS150 is under simultaneous control of cAMP-dependent protein kinase and of phosphoatase activity. Thus a rapid ACTH-dependent and cAMP-dependent protein phosphorylation in intact adrenocortical cells within steroidogenic ACTH concentrations has now been demonstrated.


Transformation by Rous sarcoma virus: effects of src gene expression on the synthesis and phosphorylation of cellular polypeptides. Infection of chicken embryo fibroblasts by Rous sarcoma virus induces a variety of alterations in cellular growth and morphology. We have used two-dimensional polyacrylamide gel electrophoresis to examine the effects of viral transformation on the pattern of synthesis and phosphorylation of cellular polypeptides. Infection by Rous sarcoma virus does not appear to induce the de novo synthesis, or the complete suppression, of any of the [35S]methionine-labeled cellular polypeptides that can be resolved with this technique; however, there are quantitative changes in a minor fraction (approximately 4%) of the [35S]methionine-labeled polypeptides. When cells labeled with [32P]orthophosphate were examined, a phosphorylated polypeptide, Mr 36,000, was detected in transformed cells; this polypeptide appears within 20 min when cells infected by a temperature-sensitive mutant of Rous sarcoma virus are shifted from the nonpermissive to the permissive temperature. Phosphorylation of the 36,000 Mr polypeptide thus represents an early event in the process of transformation, and it is possible that this polypeptide is a target for the kinase activity associated with pp60src.


Autoimmune and lymphoproliferative disease in (B6-GIX+ X 129)F1 mice: relation to naturally occurring  antibodies against murine leukemia virus-related cell surface antigens. G(IX) congeneic mouse strains, C57BL/6-G(IX) (+)(B6-G(IX) (+)) and 129-G(IX) (-), have been derived from the prototype strains, B6(G(IX) (-)) and 129(G(IX) (+)). The hybrids, (B6-G(IX) (+) x 129)F(1) (G(IX) (+)F(1)) and (B6 x 129-G(IX) (-))F(1) (G(IX) (-)F(1)), differ only in regard to genetic loci controlling G(IX) antigen expression. G(IX) (+)F(1) mice spontaneously produce G(IX) antibody and often show signs of autoimmune disease and lymphoproliferative disease. G(IX) (-)F(1) mice and mice of the two parental strains (B6-G(IX) (+) and 129) of G(IX) (+)F(1) do not produce G(IX) antibody and seldom show signs of these diseases. G((ERLD)), and G((RADA1)), antibodies, natural thymocytotoxic autoantibody, and antinuclear antibodies were produced by G(IX) (+)F(1) mice. However, these four antibodies were also found in the other strains. G(IX) (+)F(1) mice develop pronounced diffuse glomerulonephritis similar to that found in systemic lupus erythematosus in man. Incidence studies in which mice were examined according to age rather than state of health showed that the lesions occurred in 38% of G(IX) (+)F(1) mice but not in G(IX) (-)F(1), B6-G(IX) (+), or 129 mice. Lymphoproliferative lesions were either reticulum cell sarcoma (RCS) type A or reactive lymphoid hyperplasia (RLH). RCS occurred more often in G(IX) (+)F(1) (38%) than in G(IX) (-)F(1) (12%) or B6-G(IX) (+) (8%). No RCS occurred in mice of the 129 strain. RLH occurred in G(IX) (+)F(1) mice (10%) but not in the other strains. From these results, the following conclusions are drawn: (i) Severe glomerulonephritis and the increased occurrence of lymphoproliferative lesions in these animals depend on the presence of G(IX) antigen; (ii) besides genes controlling G(IX) antigen expression, other genes from both parental strains are required to create the basis in the progeny F(1) mice for the development of these diseases; and (iii) the chronic production of G(IX) antibody may be necessary for the development of the severe glomerulonephritis and for the increased occurrence of lymphoproliferative diseases in G(IX) (+)F(1) mice.


Enzyme therapy in Fabry disease: differential in vivo plasma clearance and metabolic effectiveness of plasma and splenic alpha-galactosidase A isozymes. A pilot trial of enzyme replacement with splenic and plasma alpha-galactosidase A (alpha-D-galactosidase; alpha-D-galactoside galactohydrolase, EC 3.2.1.22) isozymes was undertaken in two brothers with Fabry disease, an X-linked glycosphingolipid storage disease. Six unentrapped doses (2000 units/kg) of each isozyme were administered intravenously to the respective recipients during a 117-day period. The circulating half-life of the splenic isozyme was about 10 min, whereas that for the plasma isozyme was approximately 70 min. No immune response was detected by skin and immunodiffusion tests or by alterations in the maximal activity or clearance kinetics for either isozyme after successive administrations. After each dose of the splenic isozyme, the concentration of the accumulated circulating substrate, trihexosylceramide (globotriaosylceramide), decreased maximally (approximately 50% of initial values) in 15 min and returned to preinfusion levels by 2-3 hr. In marked contrast, injection of the plasma isozyme decreased the circulating substrate levels 50-70% by 2-6 hr; the concentrations gradually returned to preinfusion values by 36-72 hr.


Ultrasensitive enzymatic radioimmunoassay: application to detection of cholera toxin and rotavirus. Rotavirus and enterotoxin-producing bacteria are major causes of diarrheal disease in humans. A method of rapid diagnosis, ultrasensitive enzymatic radioimmunoassay, has been developed to quantitatively detect cholera toxin and rotavirus. The method uses features of both enzyme-linked immunosorbent assay and radioimmunoassay; however, the sensitivity of the assay is 100- to 1000-fold more sensitive than the two parent assays. Ultrasensitive enzymatic radioimmunoassay should also be useful in measuring other biologically important agents such as drugs and hormones.