The role of the macrophages in Marek's disease: in vitro and in vivo studies.

Macrophages form S- and K-strain Leghorn chickens, susceptible and restant to Marek's disease (MD) respectively, were studied to determine the macrophage contribution to the dynamics of MD infection, tumorigenesis and genetic resistance to this disease. In vitro studies demonstrated that macrophages from bothstrains were similar in their responses toward JM strain of Marek's disease virus (MDV) and JM-1 tumor cells. Macrophages were observed to phagocytize JM virus, but the interiorized virus was not seen to replicate within the macrophage or induce antigenic changes of the cell membrane. Clearance of JM-1 tumor cells was by both cytolytic and phagocytic mechanism. In vivo selective suppression of macrophage functions by antimacrophage serum or trypan blue inoculations resulted in significantly elevated viral titers and increased tumorigenesis, as compared to infected, non-suppressed or non-infected control groups. Results from this study indicate that genetic susceptibility or resistance to MD, as exhibited by S- and K-strain chickens, respectively, is not controlled at the macrophage level. The role of the macrophage in MD infection appears to be specifically surveillance.


Enhancement of host cell reactivation of ultraviolet-irradiated Herpes simplex virus by caffeine, hydroxyurea and 5-bromodeoxyuridine.

Enhancement of host cell reactivation (HCR) of ultraviolet (UV)-irradiated herpes simplex virus (HSV) was demonstrated in cell cultures pretreated with caffeine, hydroxyurea, or 5-bromodeoxyuridine (BrdUrd). The effect of caffeine on HCR was shown to depend on the time of drug treatment with respect to infection. In cultures treated with caffeine during the course of virus replication, the infectivity of irradiated HSV was reduced about nine-fold, while cultures pretreated with the drug before infection showed an increase in infectivity. The extent of HCR enhancement depended on the time interval between treatment with caffeine and infection, drug concentration, and the UV irradiation dose to which HSV was exposed. Magnitude of enhancement of HCR by caffeine differed in various cell species. The results suggest that enhanced HCR of UV-irradiated HSV by DNA antimetabolities is associated with DNA repair activated in consequence of cell DNA damage.


Lymphomas resembling lymphoid leukosis in chickens inoculated with reticuloendotheliosis virus.

Chickens inoculated as embyros or at hatching with the chick syncytial strain of reticuloendotheliosis virus developed a high incidence of lymphoid neoplasms between the 17th and 43rd weeks of age, involving principally the liver and bursa of Fabricius. On the basis of organ distribution, latent period, pathology and surface IgM production, the lymphomas closely resembled those of lymphoid leukosis. One inoculated chicken developed a myxosarcoma. No tumors were observed in uninoculated controls. The tumor-bearing chickens were free of infection with Marek's disease virus and exogenous avian leukosis virus (ALV) of subgroups A, B, C or D. However, the chickens were known to express endogenous ALV genes to varying degrees.


Idiopathic mammary tumors in BALB/c mice.

A colony of BALB/c mice consisting of two sublines with a high incidence of mammary tumors was examined for the presence of a mammary tumor virus (MuMTV). The mammary tumor incidences in the two sublines were 18% and 35% at average tumor age 19-20 months. Over a period of 8 years, their milk at third to tenth lactations were monitored for the presence of MuMTV antigen,and the milk and tumors were examined for the presence of B particles. Neither antigen nor B particles were found. Milk and tumor extracts from the higher mammary tumor lines were also assayed for MuMTV bioactivity by intraperitoneal inoculation of weanling C57BL, BALB/c, and RIIIf females. No response was obtained, except possible in RIIIf. Both the MuMTV antigen incidence and the tumor incidence in inoculated RIIIf mice were somewhat elevated over controls. The question remains unanswered as to whether there is an active MuMTV in our colony of tumor-bearing BALB/c mice and, if there is, whether it is associated with B particles.


Antibodies against haptens in the study of structure and biological activity of growth hormone.

The alpha-amino group of bovine Growth Hormone was selectively modified with TNBS with no detectable changes in growth promoting activity. TNP was used as hapten for the production of antibodies against the end terminal region of bGH. The region near the alpha-amino group is not involved in the binding of bGH to rat liver cells, and it seems to be away from the part of the molecule that interacts with the cell binding sites.


Studies on pituitary follitropin. V. Isolation of the subunits of the human hormone and reaction of the amino groups with acylating agents.

The alpha and beta subunits of human follitropin were isolated in a high state of purity. The tryptophan fluorescence of the native hormone and the isolated beta subunit are different. The N-terminus of the alpha and beta subunits was identified as valine and aspartic acid respectively. While recombination of the isolated alpha and beta subunits restores the electrophoretic mobility of the intact hormone, its receptor binding activity cannot be fully regenerated. Substitution of the human follitropin alpha by an ovine lutropin alpha subunit, to form a recombinant with the follitropin beta subunit, generates a complex with 2-3 receptor binding activity of the native human follitropin and the same activity as ovine follitropin. Acylation of the intact hormone does not disrupt the quaternary structure but leads to complete inactivation. Acylation studies with the subunits suggests the crucial role of the epsilon-amino groups of the alpha subunit in determining biological activity.


Tumor regrowth after irradiation; an experimental approach.

Structural changes in irradiated tumors and their regrowth were studied in a rat hepatoma, AH109A, using histological and transparent-chamber techniques. The development of the tumour was examined by means of vascular morphometry as observed in the chamber. Schematically, the tumour tissue was divided into four isocentric layers according to vascular morphology and measurements of vessel volume, surface area, and length per mm3 of tissue. The vascularity was greatest in the outermost region, decreased towards the inner parts and reached an absence of vascularity at the central necrosis. The tumors were gamma- or X-irradiated with various doses. The inside hypoxic region was destroyed completely after 3000 rad, and regrowths started exclusively from the outermost area of the tumour where enhancement of the effect of radiation by oxygen was thought to be greatest. Possible mechanisms of tumour regrowth are discussed.


Ocular surface manifestations of the major viruses.

The ocular surface is often the first site of involvement by viral infections. Background information, clinical presentations, and current treatment of three major viruses--herpes simplex, varicella-zoster, and adenovirus, have been presented in detail in this chapter. The effects of adenoviruses are usually transient and may be regarded as a nuisance. Infection with herpes simplex or varicella-zoster may lead to prolonged activity of the disease and life-long treatment with occasional loss of useful vision. It is apparent that our understanding of the clinical behavior of the major viruses has increased in recent years. Therapeutic criteria and modalities have also improved for some of the complications of the viruses. Much work needs to be done for some of the other manifestations, i.e., failure of reepithelialization, stromal melting, scarring, vascularization, and inflammation.


Demonstration of a protease inhibitor in the cornea.

Sequential extraction of bovine corneal homogenates with aqueous 0.154M NaCl, 0.5M NaCl and 3M guanidine HCl revealed the presence, in the two sodium chloride extracts, of trypsin inhibitory factors. Upon gel-filtration chromatography of the o.5M NaCl soluble corneal material on Sephadex G-75, two peaks with trypsin inhibitory activity were resolved. One peak was eluted in the void volume, whereas a second peak had mobility corresponding to a molecular weight fraction much lower than, and therefore distinct from, alpha 1-antitrypsin inhibitor. The possible implication of this inhibitory factor in the pathogenesis of corneal ulceration is briefly discussed.


Temperature sensitivity of the arenavirus junin isolated from persistently infected Vero cells.

A persistently infected cell culture was established from Vero cells surviving primary infection with wild-type Junin virus (JVwt). The cells were refractory to superinfection by both Junin virus and the related Tacaribe virus. Replication of virus released from the persistently infected cells (JVpi) was inhibited at 39 degrees. JVpi did not interfere with JVwt at 37 degrees, and it was inactivated at 37, 40, 43, 46 and 50 degrees much more rapidly than was JVwt.


Disulfide bonds in Sendai virus glycoproteins.

The precursor of fusion protein (Fo) in Sendai virus growth in Vero cells can be cleaved by trypsin to forms F1 and F2, which can be resolved on SDS-polyacrylamide gels. However, if disulfide bonds are preserved during electrophoresis, F1 and F2 remain linked together even after trypsin treatment (F). Sendai virus growth in embryonated chicken eggs does not contain the precursor Fo. However, an F protein was found for Sendai virus grown in eggs when disulfide bonds were preserved during electrophoresis. The hemagglutinin-neuraminidase (HN) glycoproteins also appear to be disulfide-linked to form large complexes which are observed on SDS-polyacrylamide gels of nonreduced samples.


In vitro synthesis of infectious DNA copies of the Rous sarcoma virus genome.

Detergent-disrupted virions of Rous sarcoma virus can synthesize in vitro an infectious viral DNA. Infectivity of the DNA was determined in transfection assays at the endpoint dilution. It was found that the specific infectivity of the crude endogenous reaction product was 34 infectious units/microgram. The endogenous reaction yielded up to 8.5 x 10(-7) infectious units per biologically active input virion, i.e., nearly 0.1 -1 infectious molecules. The possible structure of these molecules is discussed.


[Herpesviruses in man].

The importance and the extent of the inter-actions between herpes viruses and man is beginning to be understood. As more is learned about the natural histories of these viruses, it is becoming apparent that they have in common: Horizontal spread during childhood, often with subclinical infection. Latency in the host for many years, probably for life. Potential for reactivation at a later time. Host immune response maintained for life. Potential for oncogeneiss. A more thorough knowledge of the biochemistry and the biology of these viruses promises to be the foundation of more effective treatment and prevention.


Myoclonic encephalopathy of infants or "dancing eyes syndrome". Report of 7 cases with long-term follow-up and review of the literature (cases with and without neuroblastoma).

We describe 7 children with myoclonic encephalopathy of infants (MEI). MEI is a clinical entity characterized by an acute or subacute onset of polymyoclonia, cerebellar ataxia and opsoclonus ("dancing eyes"). It occurs either spontaneously, following an infectiuos illness or in association with an occult neuroblastoma. It is likely that immunological factors play a role in the pathogenesis. Steroid therapy resulted in rapid dramatic improvement of the neurological symptoms in 4 cases. However, this initial response did not correlate with the eventual outcome. We reviewed the literature to compare 45 reported cases of MEI associated with a neuroblastoma with 48 children without such a tumor to identify possible differences in clinical presentation, response to steroid medication and long-term prognosis of the neurological syndrome. In this respect we found no differences. Impairment of motor, verbal or intellectual performance were reported in at least half the cases. Although an immediate and marked response to steroids occurs in many cases of both groups, it remains unclear whether the long-term outcome is favourably influenced by this medication. The two-year-survival rate (90%) in the neuroblastoma group and the percentage of mediastinal localisation of the tumor (49%) are much higher compared with neuroblastomas without MEI. The reasons for these remarkable differences are not known. Diagnostic, therapeutic and prognostic implications justify the separation of MEI from the more common and benign syndrome known as acute cerebellar ataxia of childhood.


Some observations on the phosphatase cytochemistry of the submandibular gland of cat.

The localization of reaction product arising from incubation with thiamine pyrophosphate at pH 7.2 and beta-glycerophosphate or nitrophenylphosphate at pH 5 has been examined in central and demilunar acinar cells, myoepithelial cells, and cells of the striated ducts. Thiamine pyrophosphate reaction product was seen in the inner saccules of the Golgi apparatuses of the different types of cell. beta-Glycerophosphate and nitrophenylphosphate reaction products were seen in lysosomes, and in GERL-like structures in all except the cells of the striated ducts, in which the reaction products were observed in the Golgi apparatus. In central cells, the latter two reactions products were also seen in secretory granules and the cytosol.


The localization and properties of membrane adenosine triphosphatases in the guinea-pig placenta.

The distribution and properties of cytochemically demonstrable phosphatases in the near-term guinea-pig placenta were examined using a strontium capture technique for sodium- and potassium-dependent adenosine triphosphatase (Na+, K+-ATPase) and a lead capture technique for magnesium-dependent adenosine triphosphatase (Mg2+-ATPase). Localizations with the strontium technique in the presence of an alkaline phosphatase inhibitor were mainly on the syncytiotrophoblast plasma membranes; the reaction was potassium-dependent and ouabain-sensitive. Reaction product using the lead capture method was found on both trophoblast and endothelial cell plasma membranes and was independent of magnesium and insensitive to p-hydroxymercuribenzoate (POHMB), an inhibitor of membrane ATPases. However, a very large proportion of this reaction could be blocked by an alkaline phosphatase inhibitor. It is concluded that the strontium capture technique gave a reliable localization for Na+, K+-ATPase. However, the lead capture method mainly demonstrated alkaline phosphatase, and does not offer a useful approach to specific ATPase studies in this particular system.


Electron spin resonance (ESR) spectrum of paraffin embedded human liver tissue. A possible method for the estimation of copper content.

Human liver tissues embedded in paraffin wax for histological examination have been studied by Electron Spin Resonance (ESR) spectroscopy. A signal was detected at g approximately 2.05 section of the spectrum. The amplitude of this signal was correlated with the copper content of the embedded specimens measured by flame atomic absorption technique. The positive correlation which has been found can make ESR spectroscopy suitable for estimating the copper content of tissues without damaging the sample. The limits and errors of this method have also been analysed.


[The treatment of glomus jugulare tumours (author's transl)].

Symptoms and signs in 25 patients with glomus jugulare tumours are described. 14 patients with advanced tumours were treated exclusively by irradiation. No case of progression was noted during a follow-up period averaging 7.9 years.


[Carotid body and glomus vagale tumours (author's transl)].

Twentyfour chemodectomas (non-chromaffin paragangliomas) of the neck seen in 20 patients are presented. The investigation including angiography and the surgery in respect to risk factors and preoperative assessment is discussed. The long term results are also presented. Special note is made of the familial incidence since two twin brothers who had bilateral tumours are included.


Hydroxyl radical formation in phagocytic cells of the rat.

Polymorphonuclear leukocytes (PMN) and macrophages, harvested from the peritoneum and lung, release superoxide (O-.2) and hydrogen peroxide (H2O2) during phagocytosis. These two agents are thought to react with each other to produce a highly active oxidative substance known as hydroxyl radical (OH.). We present evidence suggesting that these radicals are generated by phagocytic cells of the rat. Our findings are based upon an assay where ethylene gas is generated from methional by the action of this radical. Ethylene generation was shown to be inhibited by superoxide dismutase, catalase, and scavengers of OH.. Of the cells examined, PMN generated the most ethylene from methional, exhibiting a fourfold increase during phagocytosis. Pulmonary and peritoneal macrophages caused smaller amounts of this gas to be formed. Regardless of cell type, an intact cell was required for ethylene generation. Zymosan appeared to be the most effective particle for all cells in ethylene formation from methional, although opsonization was critical only for PMN. Ethylene generation was dependent on cell concentration to an extent and increased with time.


Longitudinal mixing in pulmonary airways: comparison of inspiration and expiration.

The increase in dispersion of an inert tracer bolus of helium (He) or sulfur hexafluoride (SF6) was used as a direct and noninvasive measure of longitudinal mixing in the conducting airways. Over a 0- to 260-ml range of bolus penetration, a change in the inspiratory flow from 0.35 to 1.2 l/s did not affect the dispersion of He and SF6, this implies that pure convective dispersion is the dominant inspiratory mixing process. The same change in expiratory flow caused a significant increase in SF6 dispersion only; this indicates that Taylor dispersion is important during expiration. In experiments performed at a fixed penetration of 160 ml and with SF6 only, the inspiratory and the expiratory flows were independently varied from 0.18 to 5.4 l/s. Though the bolus dispersion exhibited a mildly negative correlation with inspiratory flow, it was linearly correlated with expiratory flow. From the former result we conclude that although inspiratory mixing occurs primarily by convective dispersion, there is also a small degree of turbulent dispersion. The latter result confirms that expiratory mixing is primarily due to Taylor dispersion.


Glycogen, hyaluronate, and some other polysaccharides greatly enhance the formation of exolipase by Serratia marcescens.

Among 21 different polysaccharides tested, 5 greatly enhanced the spontaneous and cyclic AMP-induced formation of exolipase: glycogen, hyaluronate, laminarin, pectin B, and gum arabic. These polysaccharides have in common the tendency to form highly ordered networks because of the branching or helical arrangement, or both, of their molecules. None of the polysaccharides could be utilized by the cells as the sole carbon source. Strong lipid extraction of four different polysaccharides did not reduce their exolipase-enhancing efficacy. At a constant cell density the stimulation of exolipase formation by various concentrations of glycogen followed saturation kinetics, suggesting a limited number of "sites" for the glycogen to act. The active principle present in a solution of pectin was destroyed by degradation (beta-elimination) of the polymer. Hyaluronate lost its exolipase-enhancing activity by exhaustive hydrolysis with hyaluronidase but was resistant to proteinase K. Exopolysaccharide, isolated from growth medium of Serratia marcescens SM-6, enhanced the exolipase formation as efficiently as hyaluronate. The results of this work are discussed mainly in terms of the "detachment hypothesis."


Effects of 8-substituted analogs of cyclic adenosine 3',5'-monophosphate on in vivo and in vitro syntheses of beta-galactosidase in Escherichia coli.

Several 8-substituted alkylthio and alkylamino cyclic adenosine 3',5'-monophosphate (cAMP) derivatives were tested for their ability to stimulate beta-galactosidase synthesis in Estherichia coli in vivo and in vitro and to inhibit the cAMP phosphodiesterase activity of E. coli. Stimulation of beta-galactosidease synthesis in vivo by cAMP derivatives decreased with increasing length of the unbranched carbon chain of the substituent. On the other hand, the stimulation in vitro was increased as the carbon chain elongated. The 8-decylthio- and 8-dodecylthio-cAMP compounds stimulated beta-galactosidase synthesis almost eight-fold compared with cAMP, whereas 8-undecyl-, 8-dodectyl-, and 8-tridecylamino-cAMP stimulated beta-galactosidase synthesis about threefold. However, in in vitro experiments with a phosphodiesterase-deficient strain of E. coli, the Crooks strain, the stimulatory effects of the derivatives disappeared, except for 8-dodecylthio cAMP which stimulated beta-galactosidase about 1.4- to 1.6-fold. All derivatives were quite resistant to hydrolysis by phosphodiesterase. Most derivatives competitively inhibited the hydrolysis of cAMP by phosphodiesterase.


Chemical alterations in cell envelopes of polymyxin-resistant Pseudomonas aeruginosa isolates.

Cell envelopes from Pseudomonas aeruginosa strains resistant to polymyxin were compared with cell envelopes from polymyxin-sensitive strains as to their content of total protein, carbohydrate, and 2-keto-3-deoxyoctonate and as to their protein composition as determined by slab polyacrylamide gel electrophoresis. The cell envelopes of the polymyxin-resistant strains had reduced amounts of lipopolysaccharide, as indicated a reduction in both carbohydrate and 2-keto-3-deoxyoctonate concentrations, and a greatly altered protein composition as shown by polyacrylamide gel electrophoresis. There was a quantitative increase in total cell envelop protein in these strains. However, those protein bands identified as being major outer membrane proteins upon polyacrylamide gel electrophoresis of separated outer and cytoplasmic membranes were reduced greatly in concentration in the polymyxin-resistant cell envelopes. Thus, it appears that polymyxin resistance in these strains is associated with the alteration of the outer membrane through a loss of lipopolysaccharide and outer membrane proteins.


Yeast cytochrome c-specific protein-lysine methyltransferase: coordinate regulation with cytochrome c and activities in cyc mutants.

The cytochromes c of fungi and higher plants contain one or two residues of epsilon-N-trimethyllysine, whose biological role is unknown. A cytochrome c-specific S-adenosylmethionine:protein-sysine methyltransferase (methylase) activity was shown to be present in extracts of the bakers' yeast Saccharomyces cerevisiae, and basic kinetic properties of this enzyme are described. The specific activity of the methylase was lower in extracts of cells grown under conditions of catabolite (glucose) repression or anaerobiosis where cytochrome c levels were low, compared with cells grown under derepressed conditions where cytochrome c levels were high. During anaerobic-to-aerobic adaptation, the methylase was induced in parallel with cytochrome c, thus suggesting that the syntheses of cytochrome c and cytochrome c methylase are coordinately regulated. None of the cyc strains surveyed (cyc1, cyc2, cyc3, cyc4, cyc5, and cyc6) had diminished levels of methylase, although some of them were completely or almost completely deficient in cytochrome c.


Aromatic amino acid biosynthesis: regulation of shikimate kinase in Escherichia coli K-12.

Starvation of cells of Escherichia coli K-12 for the aromatic amino acids results in an increased rate of synthesis of shikimate kinase activity. The two controlling amino acids are tyrosine and tryptophan, and starvation for both results in derepression. The product of the regulator gene tyrR also participates in this control, and shikimate kinase synthesis was depressed in tyrR mutants. Chromatography of cell extracts on diethylaminoethyl-Sephadex allowed partial separation of two shikimate kinase enzymes and demonstrated that only one of these subject to specific repression control involving tyrR. By contrast, chromatography of cell extracts with G-75 or G-200 columns revealed a singl-molecular-weight species of shikimate kinase activity with an apparent molecular weight of 20,000. The levels of shikimate kinase in a series of partial diploid strains indicated that aroL, the structural gene for the tyrR-controlled shikimate kinase enzyme, is located on the E. coli chromosome between the structural genes proC and purE. By means of localized mutagenesis, an aroL mutant of E. coli was isolated. The mutant was an aromatic prototroph and, by the criterion of column chromatography, appeared to have only a single functional species of shikimate kinase enzyme.


Pyridine nucleotide cycle of Salmonella typhimurium: regulation of nicotinic acid phosphoribosyltransferase and nicotinamide deamidase.

Nicotinic acid phosphoribosyl transferase (NAPRTase) and nicotinamide deamidase activities from Salmonella typhimurium were examined regarding their regulation by either feedback inhibition or repression mechanisms. The results indicate that neither enzyme is subject to feedback inhbition. Nicotinamide deamidase does not appear to be under repression control. NAPRTase, however, is repressed when cells are grown in minimal medium supplemented with various intermediates of the pyridine nucleotide cycle. The concentration of exogenously supplied pyridine nucleotide necessary to effect repression of NAPRTas was found to be that concentration which will result in a nadA mutant generation time of less than 60 min. Furthermore, the results presented indicate that nicotinamide adenine dinucleotide is the actual corepressor molecule. The analogs 6-aminonicotinic acid and 6-aminonicotinamide were also capable of repressing NAPRTase, but only when an intact pyridine nucleotide cycl permitted conversion to 6-aminonicotinamide adenine dinucleotide. The role of a repressible NAPRTase is discussed in relation to the overall functioning of the pyridine nucleotide cycle.


Orientation of the guanine operon of Escherichia coli K-12 by utilizing strains containing guaB-xse and guaB-upp deletions.

Temperature induction of an Escherichia coli strains with lambda cI1857 integrated in the guaB gene has been used to produce strains containing chromosomal deletions extending into the xse and upp genes. By utilizing strains containing these deletions, it has been possible to order the genes in the guanine operon with respect to the xseA and upp genes. The order of the genes in this region is glyA-hisS-xseA-guaO-guaB-guaA-purG-upp-purC.


Uptake and catabolism of D-xylose in Salmonella typhimurium LT2.

Salmonella typhimurium LT2 grows on D-xylose as sole carbon source with a generation time of 105 to 110 min. The following activities are induced at the indicated time after the addition of the inducer, D-xylose: D-xylulokinase (5 min), D-xylose isomerase (7 to 8 min), and D-xylose transport (10 min). All other pentoses and pentitols tested failed to induce isomerase or kinase. Synthesis of D-xylose isomerase was subject to catabolite repression, which was reversed by the addition of cyclic adenosine monophosphate. Most of the radioactive counts from D-[14C]xylose were initially accumulated in the cell in the form of D-xylose or D-xylulose. D-Xylose uptake in a mutant which was deficient in D-xylose isomerase was equal to that of the wild type. The apparent Km for D-xylose uptake was 0.41 mM. Some L-arabinose was accumulated in D-xylose-induced cells, and some D-xylose was accumulated in L-arabinose-induced cells. D-Xylitol and L-arabinose competed against C-xylose uptake, but D-arabinose, D-lyxose, and L-lyxose did not. Osmotic shock reduced the uptake of D-xylose by about 50%; by equilibrium dialysis, a D-xylose-binding protein was detected in the supernatant fluid after spheroplasts were formed from D-xylose-induced cells.


Genetics and regulation of D-xylose utilization in Salmonella typhimurium LT2.

Twenty-one Xyl- mutants of Salmonella typhimurium were selected: all had lost one or more of the activities for D-xylose isomerase, C-xylulokinase, or D-xylose transport. The mutants were classified into five functional groups: xylR, pleiotropic negative (12 mutants); xylA, D-xylose isomerase defective (3 mutants); xylB, D-xylulokinase defective (2 mutants); xylT, D-xylose transport defective (1 mutant); and 3 mutants with defective D-xylose isomerase and D-xylulokinase. Some nonsense mutations were identified among the xylR mutants. Two F'xyl plasmids were isolated by selection for early transfer of xyl+ by an Hfr which transfers xyl as a terminal gene; a plasmid with a mutation in the xyl genes, F'xylR1, was also isolated. Complementation tests using F'xyl plasmids indicate that expression of the xylA, xylB, and xylT genes is under the positive control of the xylR regulatory gene. Conjugation crosses and P22-mediated transduction data indicate that all the xyl mutations tested are in a cluster of genes at 78 units on the linkage map, and that the gene order is xylT--xylR--xylB--xylA--glyS--mtlA,D.


Arylsulfatase in Salmonella typhimurium: detection and influence of carbon source and tyramine on its synthesis.

Arylsulfatase synthesis was shown to occur in Salmonella typhimurium LT2. The enzyme had a molecular weight of approximately 50,000 and was separated into five forms by isoelectrofocusing. The optimal pH for substrate hydrolysis was pH 6.7, with Michaelis constants for nitrocatechol sulfate and nitrophenyl sulfate being 4.1 and 7.9 mM, respectively. Enzyme synthesis was strongly influenced by the presence of tyramine in the growth medium. The uptake of [14C]tyramine and arylsulfatase synthesis were initiated during the second phase of a diauxie growth response, when the organism was cultured with different carbon sources. Adenosine 3',5'-cyclic monophosphoric acid enhanced the uptake of tyramine and the levels of arylsulfatase synthesized. However, the addition of glucose and glycerol to organisms actively transporting tyramine and synthesizing enzyme caused a rapid inhibition of both of these processes. This inhibition was not reversed by adding adenosine 3',5'-cyclic monophosphoric acid. The results suggest that the effect of the carbon source on tyramine transport and arylsulfatase synthesis may be explained in terms of inducer exclusion.


Neisseria gonorrhoeae membrane microenvironment studied by spin-label electron spin resonance: comparison of colony types.

Spin-label electron spin resonance was used to characterize the microenvironment around spin probes which localize (i) in membranes, (ii) at the membrane surface, or (iii) in the cytoplasm of living Neisseria gonorrhoeae. Four colony types (T1, T2, T3, and T4) of gonococci were compared on the basis of the electron spin resonance parameters 2T parallel to, S (order parameter), and tau c (microviscosity). The concentration of spin label used had little or no effect on viability. T1 and T2 gonococci were found to have a more restricted environment for molecular motion of a membrane surface spin label than did T3 and T4. The membrane fluidity, as measured by a membrane lipid spin label, of T4 (S = 0.571) was significantly greater than that of T1 or T3 (S = 0.580). This difference was detected at 37 degrees C, at 25 degrees C, in agar-grown bacteria, and in exponential-phase cells. Studies using spin labels which probe different levels of the membrane indicated the presence of a membrane flexibility gradient. Cytoplasmic spin-label studies indicated that the cytoplasm of all gonococcal colony types was three to five times more viscous than water.


Effects of cerulenin upon the syntheses of lipid and protein and upon the formation of respiratory enzymes in adapting, lipid-limited Saccharomyces cerevisiae.

When bakers' yeast cells were grown anaerobically in a medium supplemented with Tween 80 and ergosterol, exposure during aeration to the fatty acid synthesis inhibitor, cerulenin, had little effect upon respiratory adaptation, the induction of enzymes of electron transport, or the in vivo incorporation of [(14)C]leucine into mitochondrial membranes. These lipid-supplemented cells were apparently able to undergo normal respiratory adaptation utilizing endogenous lipids alone. The level of cerulenin used (2 mug/ml) inhibited the in vivo incorporation of [(14)C]acetate into mitochondrial membrane lipids by 96%. If, however, the cells were deprived of exogenous lipid during anaerobic growth, subsequent exposure to cerulenin severely reduced their capacity to undergo respiratory adaptation, to form enzymes of electron transport, and to incorporate amino acid into both total cell and mitochondrial membrane proteins. This cerulenin-mediated inhibition of enzyme formation and of protein synthesis was nearly completely reversed by the addition of exogenous lipid during the aeration of the cells. In lipid-limited cells, chloramphenicol also had dramatic inhibitory effects, both alone (75%) and together with cerulenin (85%), upon total cell and mitochondrial membrane [(14)C]leucine incorporation. This marked chloramphenicol-mediated inhibition was also largely reversed by exogenous lipid. It is concluded that, in lipid-limited cells, either cerulenin or chloramphenicol may prevent the emergence of a pattern of lipids required for normal levels of protein synthetic activity. The effect of cerulenin upon the formation of mitochondrial inner membrane enzymes thus appears to reflect a nonspecific effect of this antilipogenic antibiotic upon total cell protein synthesis.


Cardiolipin accumulation in the inner and outer membranes of Escherichia coli mutants defective in phosphatidylserine synthetase.

Mutants of Escherichia coli defective in phosphatidylserine synthetase (pss) make less phosphatidylethanolamine than normal cells, and they are temperature sensitive for growth. We have isolated a new mutant, designated RA2021, which is better than previously available strains in that the residual phosphatidylethanolamine level approaches 25% after 4 h at 42 degrees C. The total amount of phospholipid normalized to the density of the culture is about the same in RA2021 (pss-21) as in the isogenic wild-type RA2000 (pss(+)). Consequently, there is a net accumulation of polyglycerophosphatides in the mutant, particularly of cardiolipin. The addition of 10 to 20 mM MgCl(2) to a culture of RA2021 prolongs growth under nonpermissive conditions and prevents loss of cell viability, but it does not eliminate the temperature-sensitive phenotype. Divalent cations, like Mg(2+), do not correct the phospholipid composition of the mutant, but may act indirectly by balancing the negative charges of phosphatidylglycerol and cardiolipin. To determine the effects of the pss mutation on membrane composition, we have examined the subcellular distribution of the polyglycerophosphatides that accumulate in these strains. All of the excess anionic lipids of RA2021 are associated with the envelope fraction and are distributed equally between the inner and outer membranes. The protein compositions of the isolated membranes do not differ significantly in the mutant and wild type. The fatty acid composition of RA2021 is almost the same as wild type at 30 degrees C, but there is more palmitic and cyclopropane fatty acid at 42 degrees C. These results demonstrate that the modification of the polar lipid composition observed in pss mutants affects both membranes and that cardiolipin, which is not ordinarily present in large quantities, can accumulate in the outer membrane when it is overproduced by the cell. The altered polar headgroup composition of the outer membrane in pss mutants may account, in part, for their hypersensitivity to the aminoglycoside antibiotics.


Localization of dolichyl phosphate- and pyrophosphate-dependent glycosyl transfer reactions in Saccharomyces cerevisiae.

Membranes from Saccharomyces cerevisiae protoplasts were fractionated on a continuous sucrose gradient. Six bands were obtained, which contained altogether about 15% of the total cell protein. From their densitites, their behavior in the presence and absence of Mg2+ ions, and the distribution of marker enzymes, it was possible to identify fractions enriched in rough and smooth endoplasmic reticulum and in mitochondria. All glycosyl transfer reactions investigated where dolichyl phosphates served as glycosyl acceptors or where dolichyl phosphate- and pyrophosphate-activated sugars served as glycosyl donors showed the highest specific activity and up to 75% of the total activity in the endoplasmic reticulum. This was the case for the reactions involved in the formation of O-glycosidic as well as N-glycosidic linkages in yeast glycoprotein biosynthesis. Membrane fractions enriched in plasmalemma contained less than 3% of the corresponding activities.


Decreased binding of polymyxin by polymyxin-resistant mutants of Salmonella typhimurium.

Polymyxin-resistant pmrA strains were shown to absorb only about 25% of the amount of polymyxin absorbed by the corresponding polymyxin-sensitive parent strains. The lipopolysaccharide from the pmrA strains bound less polymyxin than the lipopolysaccharide from the parent strains.


Microbial oxidation of gaseous hydrocarbons. II. Hydroxylation of alkanes and epoxidation of alkenes by cell-free particulate fractions of methane-utilizing bacteria.

Cell-free particulate fractions derived from methylotrophic bacteria catalyze the oxygen- and reduced nicotinamide adenine dinucleotide-dependent epoxidation of alkenes and hydroxylation of alkanes. Evidence presented indicates that the hydroxylation and epoxidation reactions are catalyzed by the same or a similar metal-containing monooxygenase.


Antipain lethality to Escherichia coli: dependence upon cyclic adenosine 3',5'-monophosphate and its receptor protein.

Antipain kills Escherichia coli K-12 cells in an exponential manner beginning 1 h after its addition. Mutant strains, delta cya and crp, which are unable to synthesize cyclic adenosine 3',5'-monophosphate (cAMP) and the cAMP receptor protein, respectively, are not affected. Addition of cAMP (5 mM) to antipain-treated mutant strains causes killing of delta cya cells, but not crp cells. Thus the lethal effect of antipain is dependent upon cAMP and its receptor protein.


Analgesia and haloperidol: a hypothesis.

We have previously reported 10 patient histories involving various intractable pain syndromes where the administration of Haloperidol either eliminated the need for narcotic analgesics or resulted in a significant reduction in narcotic dosage. We are presently undertaking a controlled double-blind evaluation of Haloperidol as an adjunctive treatment for intractable cancer pain. Based upon the reported clinical observations, these findings are discussed from the following aspects: 1. The isomeric similarity of Haloperidol to Meperidine. 2. Dose response between Haloperidol and analgesic effect. 3. The clinical literature regarding the use of Haloperidol for the effective withdrawal or maintenance of narcotic addicts. 4. The analgesic property as it relates to the opiate receptor.


A comment on the functional specificities of cyclic AMP-dependent and cyclic GMP-dependent protein kinases.

Cyclic AMP-dependent and cyclic GMP-dependent protein kinases (protein kinases A and G, respectively) utilize the same phosphate acceptor proteins when assayed in in vitro systems. Nevertheless, protein kinase A phosphorylates preferentially free histone, whereas protein kinase G greatly favors the histone which is associated with polydeoxyribonucleotide. On the other hand, when cytoplasmic soluble substrates such as phosphorylase kinase are used, the reactions are always more favorable for protein kinase A rather than for protein kinase G. Available evidence implies that the topographic relationship between enzyme and substrate may be an important determining factor for the functional specificities of these two classes of protein kinases.


An iron-containing superoxide dismutase from Anacystis nidulans.

Superoxide dismutase (SOD) was isolated and purified from Anacystis nidulans to near electrophoretic homogeneity. The enzyme has a molecular weight of 37,500, as determined by gel filtration and SDS-gel electrophoresis. The enzyme molecule consists of two subunits of identical molecular weight. Proton-induced X-ray elemental analysis (PIXE) showed that the SOD of A. nidulans is an iron-containing enzyme; the Fe:enzyme mol ratio was found to be 1. The EPR spectra indicated that the active center contains high-spin ferric ion. Based on quantitative EPR data, we conclude that eseentially all iron ions were detected in the EPR experiments and were present in the Fe3+ active center. Effective g'-values were calculated from computer-simulated spectra and analysis of the g'-value anisotropy of the +/-3/2 Kramers doublet made the calculation of crystal field parameters possible. The symmetry of the Fe3+ ion in the SOD molecule was found to be close to rhombic (E/D=0.240).


Flavocytochrome c of Chromatium vinosum. Some enzymatic properties and subunit structure.

The function and the structural features of Chromatium vinosum cytochrome c-552 have been investigated. Cytochrome c-552 has a sulfide-cytochrome c reductase activity and also catalyzes the reduction of elementary sulfur to sulfide with reduced benzylviologen as the electron donor. In the sulfide-cytochrome reduction, horse and yeast cytochromes c act as good electron acceptors, but cytochrome c' or cytochrome c-553(550) purified from the organism does not. The subunit structure of cytochrome c-552 has been studied. The cytochrome is split by 6 M urea into cytochrome and flavoprotein moieties with molecular weights of 21,000 and 46,000, respectively. The flavoprotein moiety is obtained by isoelectric focusing in the presence of 6 M urea and 0.1% beta-mercaptoethanol, while the hemoprotein moiety is obtained by gel filtration with Sephacryl S-200 in the presence of 6 M urea and 0.1 M KCl. Neither subunit has sulfide-cytochrome c reductase activity. Attempts to reconstitute the original flavocytochrome c from the subunits have been unsuccessful.


Formation of a TBA reaction product from crown ether in the presence of KO2.

Potassium superoxide (KO2), which can be dissolved in dimethyl sulfoxide containing crown ether, has been used as a source of O2-. for superoxide reaction systems. We have found that crown ether reacts with thiobarbituric acid (TBA) in the presence of KO2 to form a red pigment, which is a well-known reaction product of lipoperoxide.


Adrenocorticotropin acutely increases adrenal polyphosphoinositides.

After adrenocorticotropin (ACTH) treatment for 15 min, adrenal levels of diphosphoinositide and triphosphoinositide appear to increase severalfold. Several other adrenal phospholipids are not appreciably affected by such treatment. Rapid changes in polyphosphoinositides may play a role in hormonal modulation of membrane-associated metabolic events.


Phosphorylation of hydroxyproline in a synthetic peptide catalyzed by cyclic AMP-dependent protein kinase.

The cyclic AMP-dependent protein kinase catalyzes the phosphorylation of hydroxyproline present in the heptapeptide, Leu-Arg-Arg-Ala-Hyp-Leu-Gly. The Km value for the reaction with this substrate was high (approximately 18 mM) compared to the Km values reported for the analogous threonine and serine-containing peptides, which were 0.59 mM and 0.016 mM, respectively (Kemp, B.E., Graves, D.J., Benjamini, E., and Krebs, E.G. (1977) J. Biol. Chem. 252, 4888-4894). The Vmax value with the hydroxyproline-containing peptide was 1 mumol . min-1 mg-1 in contrast to Vmax values of 6 mumol . min-1 mg-1 and 20 mumol . min-1 mg-1 for the threonine- and serine-containing peptides, respectively. Phosphate esterified to hydroxyproline present in the peptide was relatively stable in hot alkali, only 10% being released as Pi within 30 min in 0.1 N NaOH at 100 degrees C, whereas all of the phosphate was released from the phosphoserine peptide analogue under these conditions. Phosphohydroxyproline in the peptide was also more stable to acid (5.7 N HCl, 110 degrees C) than phosphoserine, the time for 50% release as Pi being 15 h in contrast to 6 h for the latter.


The contribution of electrostatic factors to the stabilization of the conformation of cytochrome c. Studies on the maleylated protein.

All the lysines of horse heart cytochrome c were maleylated yielding a low spin product. At room temperature and low salt concentration, this product lacked the 695 nm absorption band and showed tryptophan fluorescence and circular dichroic spectra typical of denatured cytochrome c. The 695 nm band and the native tryptophan fluorescence and circular dichroic spectra were restored by addition of salts, their effectiveness being dependent on the charge of the cation. On low salt concentration, the 695 nm band was also restored by lowering the temperature. Studies of the temperature dependence of the 695 nm band indicate that the thermal denaturation of maleylated cytochrome c occurs at temperatures 60-70 degrees C lower than in the native protein. This implies a destabilization of the native conformation by 5.6 kcal/mol; a similar value is evidenced by comparative urea denaturation studies on the native and modified proteins. The results confirm the assumption that the native conformation of cytochrome c is mostly determined by interactions involving internal residues.


The role of calcium ion as a mediator of the effects of angiotensin II, catecholamines, and vasopressin on the phosphorylation and activity of enzymes in isolated hepatocytes.

Angiotensin II, catecholamines, and vasopressin are thought to stimulate hepatic glycogenolysis and gluconeogenesis via a cyclic AMP-independent mechanism that requires calcium ion. The present study explores the possibility that angiotensin II and vasopressin control the activity of regulatory enzymes in carbohydrate metabolism through Ca2+-dependent changes in their state of phosphorylation. Intact hepatocytes labeled with [32P]PO43- were stimulated with angiotensin II, glucagon, or vasopressin and 30 to 33 phosphorylated proteins resolved from the cytoplasmic fraction of the cell by electrophoresis in sodium dodecyl sulfate polyacrylamide slab gels. Treatment of the cells with angiotensin II or vasopressin increased the phosphorylation of 10 to 12 of these cytosolic proteins without causing measurable changes in cyclic AMP-dependent protein kinase activity. Glucagon stimulated the phosphorylation of the same set of 11 to 12 proteins through a marked increase in cyclic AMP-dependent protein kinase activity. The molecular weights of three of the protein bands whose phosphorylation was increased by these hormones correspond to the subunit molecular weights of phosphorylase (Mr = 93,000), glycogen synthase (Mr = 85,000), and pyruvate kinase (Mr = 61,000). Two of these phosphoprotein bands were positively identified as phosphorylase and pyruvate kinase by affinity chromatography and immunoprecipitation, respectively. Incubation of hepatocytes in a Ca2+-free medium completely abolished the effects of angiotensin II and vasopressin on protein phosphorylation but did not alter those of glucagon. Treatment of hepatocytes with angiotensin II, glucagon, or vasopressin stimulated phosphorylase activity by 250 to 260%, inhibited glycogen synthase activity by 50%, and inhibited pyruvate kinase activity by 30 to 35% (peptides) to 70% (glucagon). The effects of angiotensin II and vasopressin on the activity of all three enzymes were completely abolished if the cells were incubated in a Ca2+-free medium while those of glucagon were not altered. The results imply that angiotensin II, catecholamines, and vasopressin control hepatic carbohydrate metabolism through a Ca2+-requiring, cyclic AMP-independent pathway that leads to the phosphorylation of important regulatory enzymes.


Properties of the oscillatory cAMP binding component of Dictyostelium discoideum cells and isolated plasma membranes.

The cAMP receptor on the surface of aggregation competent Dictyostelium discoideum cells specifically binds [3H]cAMP in an oscillatory manner with a periodicity of 2 min. The oscillatory cAMP-binding component is developmentallly regulated and has the nucleotide specificity expected for recognition of chemotactic signals. The concentration dependence of the peak amplitudes of cAMP binding exhibit an apparent threshold at 10(-8) M cAMP. The threshold concentration for cAMP binding that we measure is consistent with the concentration dependence of signal relay (cAMP secretion) and the chemotactic response. The kinetic data of binding and dissociation are very rapid, consistent with the time course of oscillations in receptor capacity (affinity). Specific binding oscillations are destroyed by heat or chymotrypsin but are insensitive to trypsin or glycosidase. A plasma membrane localization of receptor is supported by enrichment of cAMP binding in a plasma membrane preparation from differentiated cells. Receptor oscillations with a 2-min period are preserved in the membrane preparations, and the peak amplitudes are increased about 10-fold consistent with the enrichment of other plasma membrane markers. The alternating change in the receptor's binding capacity for cAMP may be the basis of the relay refractory period as well as the primary oscillator involved in the generation of postreceptor events such as stimulation of adenylate cyclase, cAMP secretion, and cellular movement, all of which have been previously shown to oscillate.


Ionic effects on adrenal steroidogenic electron transport. The role of adrenodoxin as an electron shuttle.

We have shown (Seybert, D., Lambeth, D., and Kamin, H. (1978), J. Biol. Chem. 253, 8355-8358) that, whereas the 1:1 complex between adrenodoxin reductase and adrenodoxin is the active species for cytochrome c reduction, the complex is not sufficient to allow cytochrome P-45011 beta-mediated hydroxylations;adrenodoxin in excess of reductase is required. In the present studies, reduction by NADPH of excess adrenodoxin is shown to occur at a rate sufficient to support both cytochrome P-450 11 beta-mediated hydroxylation of deoxycorticosterone, and cytochrome P-450sec-mediated side chain cleavage of cholesterol. Oxidation-reduction potential and ion effect studies indicate that the mechanism of steroidogenic electron transport involves an adrenodoxin electron "shuttle" rather than a macromolecular complex of reductase, adrenodoxin, and cytochrome. The oxidation-reduction potential of adrenodoxin is shifted about -100 mV when bound to reductase, and reduction of the iron-sulfur protein thus promotes dissociation of the complex. The rate of adrenodoxin reduction is first stimulated, then inhibited by increasing salt; the effect is ion-specific, with Ca2+ approximately Mg2+ greater than Na+ greater than NH/+. Similar ion-specific rate effects are observed for both of the cytochrome P-450-mediated hydroxylations, indicating that the same reduction mechanism is required for these reactions. Increasing salt concentrations caused dissociation of the complex; dissociation of the form of the complex containing reduced adrenodoxin occurred at lower salt concentrations than that containing oxidized adrenodoxin. The order of effectiveness of ions in causing dissociation is the same as the order for stimulation of adrenodoxin reduction, suggesting a dissociation step in the mechanism. This proposed model, together with dissociation constants for the form of the complex containing either oxidized or reduced adrenodoxin, allows accurate prediction of the salt rate effects curve. For all ions, an activity maximum is seen at the ion concentration which produces the largest molar difference between associated-oxidized and dissociated-reduced states, and the model predicts the positions of the maxima for adrenodoxin reduction, 11 beta-hydroxylation, and side chain cleavage. Thus reduction-induced dissociation of adrenodoxin from adrenodoxin reductase appears to be a required step in steroidogenic electron transport by this system, and a role for adrenodoxin as a mobile electron shuttle is proposed.


Accessibility of some regions of DNA in chromatin (chicken erythrocytes) to single strand-specific nucleases.

The susceptibility of the DNA in chromatin to single strand-specific nucleases was examined using nuclease P1, mung bean nuclease, and venom phosphodiesterase. A stage in the reaction exists where the size range of the solubilized products is similar for each of the three nucleases and is nearly independent of incubation time. During this stage, the chromatin fragments sediment in the range of 30 to 100 S and contain duplex DNA ranging from 1 to 10 million daltons. Starting with chromatin depleted of histones H1 and H5 similar fragments are generated. In both cases these nucleoprotein fragments are reduced to nucleosomes and their multimers by micrococcal nuclease. Thus, chromatin contains a limited number of DNA sites which are susceptible to single strand-specific nucleases. These sites occur at intervals of 8 to 80 nucleosomes and are distributed throughout the chromatin. Nucleosome monomers, dimers, or trimers were not observed at any stage of single strand-specific nuclease digestion of nuclei, H1- and H5-depleted chromatin, or micrococcal nuclease-generated oligonucleosomes. Each of the three nucleases converted mononucleosomes (approximately 160 base pairs) to nucleosome cores (approximately 140 base pairs) probably by exonucleolytic action that was facilitated by the prior removal of H1 and H5. The minichromosome of SV40 is highly resistant to digestion by nuclease P1.


Control of membrane lipid synthesis in Escherichia coli during growth and during the stringent response.

The regulation of phospholipid synthesis in cells of Escherichia coli was studied in vivo during growth and during the stringent response to amino acid starvation. Strains harboring the hybrid plasmid pLC44-14 (Clark, L., and Carbon, J. (1976) Cell 9,  91-99), which had increased levels of glycerophosphate acyltransferase, were used to study the involvement of this enzyme in the control of phospholipid synthesis. In addition, regulation was studied by measuring the levels of three early intermediates of phospholipid synthesis:phosphatidic acid, CDP-diglyceride, and dCDP-diglyceride. The liponucleotides were measured by a new enzymatic method which allows determinations to be made on crude lipid extracts. Results from experiments on growing cells are consistent with regulation of membrane lipid synthesis occurring in fatty acid synthesis or at the level of glycerophosphate acylation, but not at any later step. Experiments on the inhibition of lipid synthesis during the stringent response make it possible to rule out explanations which involve the inhibition of a single enzyme; enzymes both before and after the liponucleotides in phospholipid synthesis must be affected.


The effects of radiation sterilization on the properties of ultrahigh molecular weight polyethylene.

The effects of radiation sterilization on ultrahigh molecular weight polyethylene were explored by freeze fractures, electron spin resonance, absorption of aqueous media, density measurements, small-angle and wide-angle x-ray scatter, differential scanning calorimetry, infrared and ultraviolet-visible spectrometry, static and dynamic mechanical fatigue measurements, and standard environmental stress cracking measurements. The rate of fluid absorption increased significantly due to the appearance of carbonyl groups. As the fluids were absorbed, density increased and small-angle x-ray scatter (SAXS) intensity diminished slightly, as expected. Small changes in mechanical properties (e.g., tensile properties and dynamic spectrum) were always consistent with the cross-linking which occurs after irradiation; the only significant mechanical deterioration was in the fatigue properties. The nonspherulitic, inhibited crystallinity which characterized this material and which is responsible for its excellent static resistance to environmental stress cracking was not at all changed.


Acute renal failure after total hip replacement.

Eight of forty-one patients undergoing total hip replacement experienced acute but not fatal renal failure postoperatively. All forty-one patients received transfusions of frozen blood and albumin and their wounds were irrigated with a bacitracin-neomycin-polymyxin solution. All subsequently had some relative hypotension. None of a second, prospective group of fifty-five patients who received the transfusion of albumin, but not frozen blood or the bacitracin-neomycin-polymyxin irrigant, had renal failure. The incidence of hypotension in this group was comparable to that in the first group, yet no cases of renal failure were seen. We therefore recommend that the combination of frozen blood and potentially nephrotoxic drugs be avoided in patients undergoing total hip replacement.


Cyclic 3',5' AMP relay in Dictyostelium discoideum. I. A technique to monitor responses to controlled stimuli.

A perfusion technique was developed to deliver [14C]adenosine 3',5'-cyclic monophosphate (cAMP) stimuli of well-defined magnitude and duration to tritium-labeled Dictyostelium discoideum amoebae and simultaneously monitor the elicited secretion of [3H]cAMP (i.e., the relay response). The tritiated compounds secreted in response to [14C]cAMP stimuli were highly enriched in [3H]cAMP and reflected an increase in intracellular cAMP accompanying stimulation rather than the release of a preexisting store or bulk cellular contents. The secretory response (per 10(6) cells) to 2-min stimuli increased during differentiation from about 0.2 pmol at 0.5 h to approximately 5 pmol of cAMP at 7 h. Without adequate perfusion, amoebae altered the level of cAMP in their environment in two ways: phosphodiesterases destroyed cAMP stimuli under some conditions so as to attenuate the relay response; under other circumstances, secreted cAMP magnified minimal exogenous stimuli into maximal responses. Amoebae, furthermore, would respond to their basal secretion of cAMP autocatalytically if its removal or destruction were interrupted. The perfusion system minimized these cell-induced modifications, allowing control of the level of the stimulus and response in quantitative studies.


Cyclic 3',5' AMP relay in Dictyostelium discoideum. II. Requirements for the initiation and termination of the response.

The secretion of 3H-cyclic adenosine 3',5'-monophosphate (cAMP) by prelabeled and suitably differentiated Dictyostelium discoideum amoebae was elicited in a perfusion apparatus by 10(-10) to 10(-5) M [14C]cAMP stimuli of defined magnitude and duration. Exogenous stimuli evoked an immediate increase in the rate of [3H]cAMP secretion which accelerated continuously to reach a peak of up to 100 times the unstimulated rate after 2--3 min of stimulation. Withdrawal of the stimulus at any time during the response led to a rapid decline to basal levels. Furthermore, a spontaneous decline in secretion rate was observed during prolonged cAMP stimulation, with a return to basal levels after 3--8 min of stimulation. After the initial secretory event, cells did not respond further to the continued presence of external [14C]cAMP unless (a) it was interrupted by a brief recovery period or (b) the level of the stimulus was increased sufficiently. Since the second increment could follow the first at any time, continuous secretion of [3H]cAMP could be sustained for up to 30 min by progressively increasing the stimulus between 10(-10) and 10(-5) M cAMP. The total magnitude of spontaneously terminated responses depended on the size of the increment in applied cAMP, larger stimuli evoking both a more rapid acceleration and a slower deceleration in [3H]cAMP secretion rate. The integrated response to a given increment in stimulus level was apparently independent of its "shape" - i.e., the duration, magnitude, and number of sub-steps in the increment. These data support two mechanistic inferences: that amoebae respond in proportion to relative increases in extracellular cAMP concentration, but adapt to the concentration of cAMP itself. The data further suggest that the initiation and termination of the response are mediated by cellular component(s) beyond cAMP-occupied receptors.


Transmembrane biogenesis of the vesicular stomatitis virus glycoprotein.

Previous work has shown that the mRNA encoding the vesicular stomatitis virus (VSV) glycoprotein (G) is bound to the rough endoplasmic reticulum (RER) and that newly made G protein is localized to the RER. In this paper, we have investigated the topology and processing of the newly synthesized G protein in microsomal vesicles. G was labeled with [35S]methionine ([35S]met), either by pulse-labeling infected cells or by allowing membrane-bound polysomes containing nascent G polipeptides to complete G synthesis in vitro. In either case, digestion of microsomal vesicles with any of several proteases removes approximately 5% (30 amino acids) from each G molecule. These proteases will digest the entire G protein if detergents are present during digestion. Using the method of Dintzis (1961, Proc. Natl. Acad. Sci. U. S. A. 47:247--261) to order tryptic peptides (8), we show that peptides lost from G protein by protease treatment of closed vesicles are derived from the carboxyterminus of the molecule. The newly made VSV G in microsomal membranes is glycosylated. If carbohydrate is removed by glycosidases, the resultant peptide migrates more rapidly on polyacrylamide gels than the unglycosylated, G0, form synthesized in cell-free systems in the absence of membranes. We infer that some proteolytic cleavage of the polypeptide backbone is associated with membrane insertion of G. Further, our findings demonstrate that, soon after synthesis, G is found in a transmembrane, asymmetric orientation in microsomal membranes, with its carboxyterminus exposed to the extracisternal, or cytoplasmic, face of the vesicles, and with most or all of its amino-terminal peptides and its carbohydrate sequestered within the bilayer and lumen of the microsomes.


Calcium transport and exchange in mouse 3T3 and SV40-3T3 cells.

The kinetics of Ca++ uptake have been evaluated in 3T3 and SV40-3T3 mouse cells. The data reveal at least two exchangeable cellular compartments in the 3T3 and SV40-3T3 cell over a 50-min exposure to 45Ca++. A rapidly exchanging compartment may represent surface-membrane-localized Ca++ whereas a more slowly exchanging compartment is presumably intracellular. The transition of the 3T3 cell from exponential growth (at 3 day's incubation) to quiescence (at 7 days) is characterized by a 7.5-fold increase in the size of the fast component. Quiescence of the 3T3 cell is also characterized by a 3.2-fold increase in the unidirectional Ca++ influx into the slowly exchanging compartment and a 3.6-fold increase in its size. The increase in size of the slow compartment at quiescence may result from a redistribution of intracellular Ca++ to a more readily exchangeable compartment, possibly reflecting a release of previously bound Ca++. In contrast, no significant change in any of these parameters is observed in the proliferatively active SV40-3T3 cells after corresponding period of incubation, even though these cells attained higher growth densities and underwent postconfluence.


Retention of differentiated properties in an established dog kidney epithelial cell line (MDCK).

Madin-Darby canine kidney (MDCK) cells grown in tissue culture have the morphological properties of distal tubular epithelial cells, form tight junctions, and lack several proximal tubular enzyme markers. Adenylate cyclase in these cells was stimulated by vasopressin, oxytocin, prostaglandins E1 and E2, glucagon, and cholera toxin. Hormone-stimulated adenylate cyclase activity in isolated membrane preparations was dependent on low concentrations of GTP and had the MgCl2 and pH optima expected for the kidney enzyme. The results, as well as the demonstration of enhanced hemicyst formation induced by cyclic AMP, suggest that the MDCK cell line has retained the differentiated properties of the kidney epithelial cell of origin. When MDCK cells were injected into baby nude mice, continuous nodule growth was observed until adulthood was attained. Histological studies revealed the presence of two cell types: normal mouse fibroblasts which comprise 80--90% of the solid nodule mass, and MDCK cells, which formed epithelial sheets lining internal fluid-filled glands. Electron microscope analysis showed that the mucosal surfaces of the cells were characterized by microvilli which faced the lumen of the glands, that adjacent MDCK cells were joined by tight junctions, and that the serosal surfaces of the epithelial sheets were characterized by smooth plasma membranes which were lined by a continuous basement membrane. These observations lead to the conclusion that the MDCK cells retain regional differentiation of their plasma membranes and the ability to regenerate kidney tubule-like structures in vivo.


Regulation of epidermal growth factor (EGF) receptor activity during the modulation of protein synthesis.

The capacity of cultured human fibroblasts to bind 125I-labeled epidermal growth factor (EGF) was measured during protein synthesis inhibition and reinitiation. Protein synthesis was inhibited by incubation of human fibroblasts in histidine-free medium supplemented with L-histidinol to produce a stringent amino acid starvation. Under these conditions 125 I-EGF binding activity decreased with a half-life of 14.5 hours. Protein synthesis could be rapidly reinitiated by the addition of L-histidine to human fibroblasts which had been preincubated in histidinol containing media for 36 to 48 hours. 125I-EGF binding activity rapidly increased upon the reinitiation of protein synthesis. In the presence of serum 100% of the original binding capacity was recovered ten hours after the reinitiation or protein synthesis, while 70% of the binding capacity was recovered in 12 hours in serum-free media. The recovery of 125I-EGF binding activity after the reinitiation of protein synthesis, was not blocked by the presence of Actinomycin D, indicating that the messenger RNA for the EGF receptor may accumulate during the period of histidinol-mediated inhibition of protein synthesis. The time course of recovery of 125I-EGF binding activity after the reinitiation of protein synthesis is very similar to that observed during the recovery of receptor activity following "down regulation" of EGF receptor activity. Recovery from down regulation, however, was markedly sensitive to Actinomycin D.


Abnormal regulation of de novo purine synthesis and purine salvage in a cultured mouse T-cell lymphoma mutant partially deficient in adenylosuccinate synthetase.

The isolation and characterization of a mutant murine T-cell lymphoma (S49) with altered purine metabolism is described. This mutant, AU-100, was isolated from a mutagenized population of S49 cells by virtue of its resistance to 0.1 mM 6-azauridine in semisolid agarose. The AU-100 cells are resistant to adenosine mediated cytotoxicity but are extraordinarily sensitive to killing by guanosine. High performance liquid chromatography of AU-100 cell extracts has demonstrated that intracellular levels of GTP, IMP, and GMP are all elevated about 3-fold over those levels found in wild type cells. The AU-100 cells also contain an elevated intracellular level of pyrophosphoribosylphosphate (PPriboseP), which as in wild type cells is diminished by incubation of AU-100 cells with adenosine. However AU-100 cells synthesize purines de novo at a rate less than 35% of that found in wild type cells. In other growth rate experiments, the AU-100 cell line was shown to be resistant to 6-thioguanine and 6-mercaptopurine. Levels of hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) measured in AU-100 cell extracts, however, are 50-66% greater than those levels of HGPRTase found in wild type cell extracts. Nevertheless this mutant S49 cell line cannot efficiently incorporate labeled hypoxanthine into nucleotides since the salvage enzyme HGPRTase is inhibited in vivo. The AU-100 cell line was found to be 80% deficient in adenylosuccinate synthetase, but these cells are not auxotrophic for adenosine or other purines. The significant alterations in the control of purine de novo and salvage metabolism caused by the defect in adenylosuccinate synthetase are mediated by the resulting increased levels of guanosine nucleotides.


Amino acid transport in normal and Rous sarcoma virus-transformed chicken embryo fibroblasts.

A study was made of the transport of a variety of amino acids by uninfected and Rous sarcoma virus-infected chicken embryo fibroblasts. Following a period of amino acid starvation, transformed, but not normal cells, showed increased levels of transport for alpha-aminoisobutyric acid, proline and alanine, three amino acids which are transported primarily by the A transport system. There was no starvation-induced increase in the transport of leucine, phenylalanine, lysine, or cycloleucine. In the absence of starvation, normal and transformed cells exhibited comparable rates of amino acid transport. Cycloheximide was able to block the increase in uptake. The enhanced uptake was characterized by an increase in Vmax for transport and little change in Km. The data demonstrate that an alteration in the regulation of the A amino acid transport system is an early event in malignant transformation by Rous sarcoma virus. However, since this alteration in made manifest only following a period of starvation, our findings suggest that increased amino acid uptake does not play a role in generating the other manifestations of the transformed state seen in cell culture.


Gonadotropin stimulation of cyclic AMP levels in Chinese hamster ovary cells in culture.

When Chinese Hamster Ovary (CHO) cells, incubated in serum-free medium, are exposed to gonadotropins a transient increase in the intracellular concentration of cyclic AMP is observed. Maximum accumulation of cyclic AMP is noted 30 minutes after addition of either human chorionic gonadotropin (hCG) or follicle stimulating hormone (FSH). Within one to two hours after hormone addition, the intracellular concentrations of cyclic AMP have returned to basal levels. The enhancement of intracellular cyclic AMP levels by hCG is hormone concentration dependent, with maximal stimulation observed at 10 micrograms/ml hCG. The exogenous addition of gonadotropins also slows the growth rate of CHO cells. This effect on growth seems to be mediated through cyclic AMP since the growth rate of a mutant of CHO cells defective in the catalytic subunit of cyclic AMP dependent protein kinase is only slightly decreased.


T98G: an anchorage-independent human tumor cell line that exhibits stationary phase G1 arrest in vitro.

T98 and T98G are two related cell lines that were derived from a human glioblastoma multiforma tumor. T98G has almost twice as many chromosomes as T98, suggesting that it is a polyploid variant of T98. Three aspects of control of cellular proliferation were studied in T98 and T98G cells in comparison to WI-38 normal human diploid cells. WI-38 cells have the following properties: (1) they can undergo only a limited number of population doublings in vitro; (2) they cannot proliferate without anchorage; and (3) they become arrested in G1 phase under stationary phase conditions. T98 cells differ from normal cells in all three of these properties, as do many other transformed cell lines. However, the derivative of T98, namely T98G, expresses an unique combination of normal and transformed aspects of the control of cellular proliferation. T98G cells are like normal cells in that they become arrested in G1 phase under stationary phase conditions, yet they also exhibit the transformed characteristics of anchorage independence and immortality. Thus, T98G cells demonstrate that transformation to immortality and anchorage independence can exist without concomitant loss of the normal mechanism for G1 arrest in response to stationary phase conditions. This result supports the hypothesis that each of these three aspects of control of cellular proliferation can be altered independently. Partially transformed cell lines, such as T98G, should be useful for sorting out the biochemical changes associated with transformation in each of these aspects.


The effects of Clostridium perfringens enterotoxin on morphology, viability, and macromolecular synthesis in Vero cells.

Vero (African green monkey kidney) cells grown in tissue culture monolayer were sensitive to Clostridium perfringens enterotoxin. Within 30 minutes of exposure to the enterotoxin gross morphological damage was observed and within 40 minutes approximately 75% of the cells had detached. Nearly half of the cells were nonviable following 35 to 40 minutes incubation with the enterotoxin. Doses as low as 0.1 ng caused small but detectable inhibition of plating efficiency of the cells while more than 100 ng caused the inhibition to approach 100%. Total inhibition of DNA, RNA, and protein synthesis occurred within 30 minutes exposure to enterotoxin. Heat inactivated enterotoxin had no apparent effects upon cellular morphology, detachment, viability, plating efficiency, or incorporation. We propose that the enterotoxin induces structural damage to the cytoplasmic membrane which results in loss of electrolytes and other essential substances from the cells. The outcome of this process is shut down of macromolecular synthesis, gross morphological damage, and eventual death of the cell.


Sulfated mucopolysaccharides from normal and virus transformed rodent fibroblasts.

The sulfated mucopolysaccharide composition of normal and virus transformed Balb 3T3 and BHK21 cell lines is reported. It is shown that normal 3T3 cells contain mainly chondroitin sulfate B and heparitin sulfate. Relatively higher amounts of chondroitin sulface AC were observed in polyoma virus transformed 3T3 cells, besides an absolute increase of all the three sulfated mucopolysaccharides in the polyoma and SV 40 transformed cells. It is shown also that the three sulfated mucopolysaccharides are at least in part at the cell surface. Similar differences in sulfated mucopolysaccharide composition of normal and virus transformed BHK cell lines were also observed.


Electrophysiology of differentiating mouse spermatozoa.

The morphological aspects of spermatogenesis are well described in many mammalian species, but functional changes are not completely understood. Electrophysiological parameters were investigated in primary spermatocytes and early and late spermatids isolated from the seminiferous tubules of the mouse. Substantial changes were not detected in membrane potential between different developmental stages. Membrane potential was dependent on both potassium and sodium ion concentration gradients, but not on chloride gradients. The ratio of the permeabilities PNa/Pk varied according to the extracellular concentrations of sodium and potassium. Ouabain, a specific inhibitor of Na+, K+-activated ATPase, produced a maximal reduction in membrane potential of 20%. Comparisons were drawn between differentiating germ cells and previously determined properties of mature spermatozoa.


Erythroid and granulocytic colony growth in cultures supplemented with human serum lipoproteins.

The ability of human serum to support erythroid and granulocytic colony formation has been investigated. It was found that normal human serum could replace fetal calf serum in the cultures and was able to support the growth of these hemopoietic colonies. Serum fractions enriched for low density lipoproteins, either by precipitation with Heparin-Mn++ or by ultracentrifugation, was found to contain this growth supporting activity of human serum.


Control of the Balb/c-3T3 cell cycle by nutrients and serum factors: analysis using platelet-derived growth factor and platelet-poor plasma.

Much controversy regarding the relationship between nutrients and serum in regulation of cell growth can be reconciled by recognizing that serum contains multiple factors which regulate different events in the cell cycle. Serum was fractionated into a platelet-derived growth factor (PDGF), which induces cells to become competent to synthesize DNA, and plasma which allows competent cells to traverse G0/G1 and enter the S phase. Nutrients are not required for the cellular response to PDGF; however amino acids are required for plasma to promote the entry of PDGF-treated, competent cells into S phase. The nutrient independent, PDGF-modulated, growth regulatory event (competence) is located 12 hours prior to the G1/S phase boundary in quiescent, density-arrested Balb/c-3T3 cells. The nutrient dependent, plasma-modulated event is located six hours prior to the G1/S phase boundary and corresponds in concentration of amino acids required for DNA synthesis. Infection of density-arrested Balb/c3T3 cells with SV40 overrides both the nutrient independent and the nutrient dependent growth regulatory events.


Properties of Na-K pump in primary cultures of kidney cells.

Activities related to Na-K transport were measured in cell cultures of ground squirrel kidney cortex in order to compare these cells with those of intact kidney and of continuous cell lines. A microsomal preparation containing plasma membrane Na,K-ATPase from fresh kidney showed twice the activity of a similar preparation from 72-hour cultured cells. Na,K-ATPase of homogenates of 72-hour cells showed one-third to one-fourth the specific activity of that from 6-hour cultured cells. The associated K-dependent phosphatase activity also declined as a function of time in culture. The ouabain-sensitive influx of K into 6-hour cultured cells was twice as great as the K influx into 72-hour cells. The number of sites binding 3H-ouabain in intact cultured cells declined 81% on a cell protein basis between 6 and 72 hours in culture. This decline in ouabain binding sites was relatively greater than that of K influx, so that the K turnover number increased over this same time period. The decline in ouabain-sensitive K influx during culture was complementary to an increase in furosemide-sensitive K influx. Measurements of unidirectional and net K fluxes showed that there were three components of K influx into 3-day cultured cells: ouabain-sensitive Na:K exchange, furosemide-sensitive K:K exchange, and K diffusion. In the 6-hour cultures, however, there was no furosemide-sensitive K:K exchange. Thus, after three days in culture ground squirrel kidney cells lose a feature characteristic of the original parent cells (high Na,K-ATPase activity), and gain a feature common to many undifferentiated cultured cells (furosemide-sensitive K:K exchange).


Tyrosinase maturation and pigment expression in B16 melanoma: relation to theophylline treatment and intracellular cyclic AMP.

We have studied the effects of theophylline treatment on pigmentation characteristics and growth of two B16 melanoma cell lines, HFH-18 and P/140. Cell counts of control and theophylline-treated cultures confirmed that the drug inhibits cell growth. Light and electron microscope cytochemistry with the L-dopa reaction indicated that the two cell lines differ in their ability to transfer Golgi-associated tyrosinase to developing premelanosomes. The results of these experiments, considered with results of electrophoretic analyses and activity measurements by the Pomerantz method, also provide evidence that increased tyrosinase synthesis occurs in response to theophylline treatment. In addition, results indicate that theophylline induces changes in the rate of synthetic or degradative posttranslational modification of tyrosinase. Measurements of intracellular cyclic AMP levels by radioimmunoassay in control cultures and in theophylline- and alpha-MSH-treated cultures were made. Although the hormone induced spectacular increases in cyclic AMP levels, theophylline produced no detectable change. These results indicate that theophylline differs from alpha-MSH because theophylline-induced changes in pigmentation may not require the participation of intracellular cyclic AMP.


Tumorigenicity of cells transformed by Simian virus 40 and of hybrids between such cells and normal diploid cells.

A number of newly isolated clonal cell lines derived from diploid mouse embryo cells transformed by SV40 were examined in vitro and in vivo. Although these lines showed the properties that define transformation in vitro, they were not tumorigenic for many passages after their initial isolation. Cells from tumours eventually produced by the SV40-transformed cells were fused with diploid mouse embryo cells. The hybrids formed were initially non-tumorigenic. This indicates that a normal diploid cell can suppress the malignant phenotype of a tumorigenic SV40-transformed cell. The hybrid cells did, however, express the SV40 T antigen and they nad a clearly transformed phenotype in vitro. It thus appears that neither the transformed phenotype nor the expression of the SV40 T antigen are enough to endow a cell with the ability to grow progressively in vivo. The relationship between the transformed phenotype and tumorigenicity was further studied by fusing malignant mouse melanoma cells with non-tumorigenic SV40-transformed cells. The hybrids expressed the transformed phenotype in vitro but unable to form tumours in vivo. The changes that occur in cells after transformation by SV40 do not apparently affect the ability of these cells to suppress the malignant phenotype of tumour cells.


The evolution of excitable behaviour in Dictyostelium.

Aggregation of Dictyostelium discoideum amoebae is effected by relayed cyclic AMP (cAMP) signals. The period of this wave propagation drops abruptly at the transition from aggregative to post-aggregative development. In this paper I demonstrate that the light-scattering response induced by a low concentration pulse of cAMP evolves from one lasting 5 min to one lasting 2 min. The definitive establishment of the 2-min response coincides with the beginning of post-aggregative gene expression. Amoebae at the aggregative stage are unable to respond to a second cAMP pulse delivered less than 4 min after the first, whereas at the post-aggregative stage they can respond to 2 pulses separated by 1 min or longer. Addition of cAMP phosphodiesterase to amoebae at the aggregative stage can in part mimic the change in excitable behaviour: the response is shortened and the amoebae can respond more frequently. However, the shortened response is not post-aggregative in shape and the prolonged presence of cyclic nucleotides induces different responses at the aggregative and post-aggregative stage. Both these results suggest that the rate of destruction of the relayed signal is not solely responsible for the change in excitability.


Studies of membrane fusion. IV. Fusion of HeLa cells with Sendai virus.

The Sendai virus-induced fusion of HeLa cells has been studied by freeze-fracture electron microscopy. Freeze-fracture observations confirm previous scanning electron-microscope studies (1977) and show that at 4 degrees C virus particles bind to the cell surface and that cell agglutination results from the crosslinking by virus particles of microvilli on adjacent cells. Incubation at 37 degrees C initiates a change in viral envelope structure and fusion of 'altered' virus particles with the cell plasma membrane. Fusion of a virus particle with two crosslinked cells is probably the membrane fusion event which initiates cell-cell fusion; fusion is completed as a result of virally induced cell swelling. Lateral diffusion of viral envelope components following virus-cell fusion and, in some instances, an aggregation of plasma membrane intramembrane particles occurs in swollen cells. These observations show that the mechanisms of viral envelope-cell and probably cell-cell fusion are the same as have been reported for erythrocytes. Although endocytosis of intact virus particles does occur, the specialized cell-mediated mechanism for fusion of the viral envelope with the cell plasma membrane suggests that this, and not viropexis, is the mechanism of Sendai virus infection.


Studies of membrane fusion. V. Fusion of erythrocytes with non-haemolytic Sendai virus.

The fusion of human erythrocytes with non-haemolytic '1-day' Sendai virus has been studied by electron microscopy. The mechanism of viral envelope-cell fusion is the same as that described previously for haemolytic '3-day' Sendai virus except that fusion is frequently arrested at an initial stage when 2 segments of smooth linear viral membrane fuse and become incorporated into the erythrocyte membrane. After longer periods of incubation at 37 degrees C, in addition to many partly fused virus particles, long (up to 4 micrometer) lengths of smooth linear viral membrane are seen within the erythrocyte membrane which arise by linear aggregation of shorter (approximately 0.25 micrometer long) segments of smooth linear membrane derived from individual fused viral envelopes. Cell-Cell fusion, as a result of the fusion of a viral envelope with 2 adjacent erythrocytes also occurs but, in the absence of cell swelling, fusion is arrested at this stage with cells joined by one (or more) small cytoplasmic bridges. Typical fused cells are produced if such cells are swollen with hypotonic buffer. These observations provide further evidence that membrane fusion and cell swelling are distinct events in cell fusion and that cell swelling is the driving force both for completing the incorporation of the viral envelope into the cell membrane and for expanding cells connected by small cytoplasmic bridges to form spherical fused cells. Little lateral diffusion of viral envelope components occurs in the absence of cell swelling; in fact, some aggregation of components occurs. Comparison with previous studies using haemolytic '3-day' Sendai virus suggests that virally induced cell swelling perturbs membrane structure so as to allow the rapid lateral diffusion of integrated viral envelope components.


Probable pituitary adenoma with adrenocorticotropin hypersecretion (corticotropinoma) secondary to Addison's disease.

A 50-yr-old woman with Addison's disease from the age of 14 yr was diagnosed as empty sella turcica in 1974 (Rev Clin Esp 139: 183, 1975). She subsequently continued with hyperpigmentation in spite of adequate hormone substitution therapy which permitted her to lead a normal life. When studied she showed an extreme elevation of plasma ACTH (1500--2000 pg/ml), and with dexamethasone (2 and 8 mg/day) continued to have levels of 900 pg/ml. With 60 mg hydrocortisone daily, effects of overdosage were observed (swelling and Cushingoid facies) associated with depigmentation. However, she continued to manifest levels of plasma ACTH of 700 pg/ml and an absence of circadian rhythm. It seems likely that this patient represents a case of pituitary ACTH secretory adenoma (corticotropinoma) secondary to the preexisting Addison's disease. The circulating levels of other pituitary hormones were normal.


Regulation of receptor by homologous hormone enhances sensitivity and broadens scope of radioreceptor assay for human growth hormone.

In standard competitive binding assays (including radioreceptor assays) unlabeled ligand (hormone) competes with labeled ligand (hormone) for binding to a fixed number of binding (receptor) sites. Detection of the unlabeled ligand occurs when the occupancy of binding sites by the unlabeled ligand is sufficient to reduce the binding of labeled ligand. A common feature of the hormone-receptor interaction is the ability of the hormone to regulate the affinity and/or the concentration of its homologous receptor. In the present study, by exploiting the ability of human GH to regulate by negative feedback the concentration of its own receptors, we have enhanced the sensitivity of the human GH radioreceptor assay 5-fold. The ability of hormone to regulate receptor concentration and affinity affords wide opportunities to broaden the scope as well as to enhance the sensitivity of radioreceptor assays.


Circadian variation of plasma catecholamines in young and old men: relation to rapid eye movement and slow wave sleep.

Young and old healthy subjects with indwelling venous cannulae were found to undergo significant diurnal variations in plasma catecholamine levels. Both norepinephrine and epinephrine levels peaked in late morning and reached lowest values at night during sleep. Catecholamine levels were similar during slow wave and rapid eye movement sleep. While epinephrine levels were unaffected by age, norepinephrine levels were greater in older subjects by 28% during the day (at 1100 h; P less than 0.01) and by 75% at night (between 2200--0900 h; P less than 0.01). Older subjects slept less well; they had 90% less stage 4 sleep, 27% less rapid eye movement sleep, and twice as much wakefulness at night (P less than 0.05). These findings raise the possibility that this well known age effect on sleep may be related to increased sympathetic nervous system activity.


Multiple symmetric lipomatosis (Launois-Bensaude syndrome) - adipose tissue insensitivity to cyclic AMP.

Adipocytes prepared from lipomatous tissue of a patient with multiple symmetric lipomatosis have been incubated in the presence of norepinephrine, or insulin, or both. No lipolytic response to norepinephrine could be detected. However, the cellular content of cyclic AMP doubled in response to norepinephrine. Insulin reversed this. The data suggest that fat mobilization from the lipoma is impaired, and that this is due to resistance to the effect of cyclic AMP on hormone sensitive lipase.


Pleocytosis and immunoglobulin changes in cerebrospinal fluid and herpesvirus serology in patients with Guillain-Barré syndrome.

In a follow-up study of 24 patients with Guillain-Barré syndrome, 55% developed a cerebrospinal fluid (CSF) mononuclear pleocytosis, which persisted for 4 months or more. Raised index values of CSF-immunoglobulin G (IgG), CSF-IgA, and CSF-IgM, indicating synthesis of the immunoglobulin in question in the central nervous system, were found in 63, 35, and 25%, respectively. Agarose gel eletrophoresis revealed oligoclonal immunoglobulin in CSF in 21% and in both CSF and serum in another 21% of the patients. Twenty-five percent had abnormally low kappa/lambda ratios of CSF and/or serum, indicating synthesis of oligoclonal immunoglobulin, mainly of the lambda light-chain type. The inflammatory reaction in the central nervous system, as reflected by pleocytosis, immunoglobulin synthesis, and oligoclonal immunoglobulin, was not correlated to the severity or course of Guillain-Barré syndrome. A raised CSF-IgM index and oligoclonal immunoglobulin were found more often in the Guillain-Barré syndrome patients who displayed pleocytosis. All patients had or developed antibodies to Epstein-Barr virus. Three patients had serology indicating a primary infection, 11 patients had antibody changes indicating a reactivated infection, and 10 had serology indicating previous exposure. Two patients showed serological evidence for a primary cytomegalovirus infection, 2 had serology indicative of a reactivated infection, 12 had titers as caused by previous exposure, and 8 remained seronegative. Virus-specific IgM was measurable in all cases of primary infection. Neither primary or reactivated Epstein-Barr virus nor cytomegalovirus infections were obviously related to CSF pleocytosis.


Thin-layer immunoassay for determination of antibodies to herpes simplex virus.

Thin-layer immunoassay (TIA) is a simple serological technique suitable for analysis of large numbers of samples. In this study, TIA was evaluated for determination of antibodies to herpes simplex virus. Herpes simplex virus antigen used in TIA was purified from material released from virus-infected cells. The results obtained by TIA were compared with those obtained by neutralization and complement fixation tests. TIA was found to be as sensitive as the neutralization test for demonstration of herpes simplex virus antibodies. No false-negative or -positive reactions were observed. In primary herpes simplex virus-1 infections, an antibody response was demonstrated by TIA, whereas antibodies could not be demonstrated in patients with primary herpes simplex virus-2 infections.


Decreased cerebrospinal fluid cyclic adenosine 3',5'-monophosphate in bacterial meningitis.

The concentration of cyclic adenosine 3',5'-monophosphate (cAMP) in 16 cerebrospinal fluid samples from eight patients with bacterial meningitis due to several different organisms was determined. An age- and sex-matched control group of 12 patients with a variety of acute, noninfectious systemic and neurological diseases was also examined. To quantitate the amount of cAMP, a new, improved radioimmunoassay was used with the ability to measure 2.5 X 10(-15) mol of cAMP. The mean concentration of cAMP in the cerebrospinal fluid from patients with meningitis was 0.05 nM, and from patients in the control group it was 1.18 nM. The difference between these two values is statistically significant. The decreased cAMP concentration in the cerebrospinal fluid from patients with bacterial meningitis did not seem to be secondary to metabolism by bacteria or leukocytes, increased enzymatic degradation within the cerebrospinal fluid, or an artifact introduced by the collection and storage procedure. Since the concentration of cAMP in the cerebrospinal fluid is normally found to be within narrow limits and probably reflects intracellular cAMP levels, the results described in this study suggest that interference with cAMP metabolism in central nervous system tissue occurs in bacterial meningitis. This finding seems to be independent of the causative organism and might explain the pathogenesis of selected, neurological manifestations of this disease.


Comparison of indirect hemagglutination and indirect immunofluorescence tests with microneutralization tests for detection of type-specific Herpesvirus hominis antibody.

Indirect hemagglutinating and immunofluorescent antibody responses to Herpesvirus hominis types 1 and 2 were compared to neutralizing antibody responses in infected humans from whom H. hominis type 1 or 2 was isolated. The indirect immunofluorescent antibody test was shown to be the most sensitive and specific for primary human infections. The sensitivity and specificity of the indirect hemagglutination and the immunofluorescent antibody tests were shown to be equal to that of the microneutralization test among patients who had primary or recurrent H. hominis type 2 infections. It is suggested that the indirect hemagglutination test is preferable for assaying large populations for previous infection with H. hominis type 2 because it is rapid, easier to perform, and more economical. The intermediate range of titer differences (deltat) between H. hominis types 1 and 2 previously reported to be due to infections with both viruses was shown to occur in all three tests among patients with primary infections with either virus.


Production of high-titer bovine rotavirus with trypsin.

Titers of bovine rotavirus in excess of 10(9) immunofluorescent infectious units per ml of culture fluids have been produced, using trypsin treatment of the virus. Infectivity of preparations of the virus can be increased with as little as 1 ng of trypsin per ml, with maximum increases of 1 to 2 log10 with 1 microgram of trypsin per ml. The virus grows to titers in excess of 10(5) immunofluorescent units per ml in MDBK, LLC-MK2, MA-104, and HeLa cells. When MDBK cells are infected with a multiplicity of infection of 20, maximum yields of cell-associated, trypsin-enhanceable virus are obtained 4 to 8 h postinfection. Maximum yields of cell-free, trypsin-enhanceable virus are produced 16 to 20 h postinfection. The results presented here indicate that trypsin can be used to produce high-titer stocks of bovine rotavirus.


Sensitivity of enzyme-linked immunosorbent assay, complement fixation, and hemagglutination inhibition serological tests for detection of Sendai virus antibody in laboratory mice.

The enzyme-linked immunosorbent assay technique for detection of Sendai virus antibody in mice was approximately 100- and 300-fold more sensitive than the complement fixation and hemagglutination inhibition tests, respectively. The assay also permitted direct quantitative measurement of the amount of antibody on a single serum dilution rather than by the more traditional serial titration.


Assay of human interferon in Vero cells by several methods.

Four methods for the assay of human interferon in Vero cells were compared based on the inhibition of viral cytopathic effect (CPE) in tubes, the inhibition of CPE in microplates, the reduction of plaques, and the inhibition of quantitative hemadsorption. For inhibition of CPE, Sindbis virus, vesicular stomatitis virus, poliovirus type 2, and vaccinia virus were used for challenge. In the plaque reduction method, Sindbis virus, vesicular stomatitis virus, and poliovirus were employed, and Newcastle disease virus was used in the quantitative hemadsorption assay. Sindbis virus was most susceptible to interferon in those tests measuring inhibition of CPE, but vesicular stomatitis virus was as sensitive in the plaque reduction method. Highest titers of interferon were recorded in microplates, especially with Sindbis virus as the challenge agent, followed by the quantitative inhibition assay. The CPE inhibition method was the simplest, and the quantitative hemadsorption assay was the most rapid to perform. Reproducibilities, as shown by the coefficient of variation, were 15, 39, and 59% for plaque reduction, CPE inhibition in tubes, and CPE inhibition in microplates, respectively.


Immunoglobulin M anti-hepatitis A virus determination reorienting gradient centrifugation for diagnosis of Acute Hepatitis A.

The persistence of antibody to hepatitis A antigen (anti-HAV) of the immunoglobulin M (IgM) class was evaluated in 88 sera of 51 acute hepatitis A patients. IgM was separated from IgG by a 2-h reorienting sucrose gradient ultracentrifugation, and the titer of anti-HAV was determined in the IgG- and IgM-containing fractions by solid-hase radioimmunoassay. IgM anti-HAV was the predominating antibody at onset of jaundice and persisted in these patients for at least 60 days, but not longer than 115 days. The demonstration of IgM anti-HAV is therefore a valuable tool for the diagnosis of recent hepatitis A infection.