Induction of virus-neutralizing antibody by glycoproteins isolated from chicken cells infected with a herpesvirus of turkeys. The production of virus-induced proteins in chicken embryo fibroblast cells infected with a herpesvirus of turkeys was studied. It was found that glycoproteins isolated from membrane-rich fractions of infected cells by affinity chromatography using concanavalin A induced neutralizing and precipitating antibody in rabbits and chickens. After analytical electrophoresis, such isolates were found to contain three polypeptide bands of between 100 x 10(3) and 120 x 10(3) molecular weight not present in glycoprotein extracts of uninfected cells, and these polypeptides were further purified by preparative polyacrylamide gel electrophoresis. Inoculation of chickens with purified material also resulted in the production of precipitating and neutralizing antibody, showing that these high-molecular-weight polypeptides play a role in the humoral immunity to Marek's disease. Challenge of these chickens with virulent Marek's disease virus revealed that a partial protection was afforded by the inoculated glycoproteins.


Serological response of the bovine fetus to bovine viral diarrhea virus. Susceptible pregnant heifers were inoculated with bovine viral diarrhea virus at 150 days of gestation and earlier. Fetuses were surgically collected at selected times after inoculation. Serum immunoglobulins were quantitated, and the presence of specific antibodies was determined. In fetuses from heifers inoculated at 150 days, immunoglobulin M (IgM) appeared approximately 2 weeks after inoculation and was followed in 7 days by IgG1. Later IgG2 was detected in the sera of three fetuses. Serum-neutralizing and complement-fixing antibodies were first detected in a fetus taken at 206 days of gestation. Fetuses taken at later times also had specific serum antibodies. Possible explanations for the appearance of serum immunoglobulin substantially before specific bovine viral diarrhea antibodies include the viral alteration of host tissues rendering them antigenic viral activation of polyclonal B cells, and viral modulation of virus-specific lymphocytes causing specific interference with the appearance of antiviral antibodies. In one of the fetuses having IgG2, the serum also contained IgA. Placental leakage of material immunoglobulins was thought to be responsible for the presence of IgA and IgG2 in this fetus. Small quantities of IgM were found in the serum of two fetuses taken from heifers inoculated between 65 and 95 days of gestation, but specific antibodies were found in none.


Elevated titer of antibodies to Simian sarcoma virus envelope antigen (gp70) and normal response to influenza virus in untreated Danish Hodgkin's patients. Untreated Danish Hodgkin's disease (HD) patients and paired age- and sex-matched controls were tested for serum antibodies to Epstein-Barr virus (EBV), six strains of influenza virus and Simian sarcoma virus/Simian sarcoma-associated virus [SSV(SSAV)] antigens. HD sera showed significantly elevated titers against EBV and both increased incidence and mean titer of antibodies to the envelope glycoprotein of SSV(SSAV), whereas testing against the influenza viruses revealed no differences between HD and controls. A focus reduction assay demonstrated a low incidence in HD and controls of sera with neutralizing effects against SSV(SSAV) and the "baboon" C-type virus component of the human HL-23 virus complex, which seemed coupled to HL-A type W19.


Growth of diploid, Epstein-Barr virus-carrying human lymphoblastoid cell lines heterotransplanted into nude mice under immunologically privileged conditions. Human Epstein-Barr virus-carrying lymphoid cell lines which have been classified on the basis of studies on clonality and morphological, chromosomal and functional parameters as lymphoblastoid cell lines (LCL) of presumed non-neoplastic origin were inoculated intracerebrally into nude mice. All eighteen of them grew, killing the host mice within 7 to 25 days, except for 2 which grew more slowly. At autopsy, the brain of the nudes was found to be invaded by infiltrating lymphomas. Sixteen of these lymphomas, when recultured in vitro, gave rise to cell lines with growth properties and morphology indistinguishable from those of the inoculated LCL. Chromosomal examinations showed that 3/7 cell lines injected, which grew as lymphomas in the brain, were still normal diploid on reexplantation whereas the remaining four had become aneuploid. Four lines derived from intracerebral lymphomas (2 diploid, 1 aneuploid and 1 untested) were inoculated subcutaneously into adult nude mice. None of them grew. When the corresponding four original LCL lines were inoculated subcutaneously into newborn nude mice, they grew rapidly, but failed to do so in newborn normal mice or intracerebrally in adult normal mice. One such line, U-1450, was treated with anti-lymphocyte serum (ALS). Small nodules developed at the site of inoculation. From one nodule a cell line was cultured, 1450 ALSAD. It was morphologically indistinguishable from the line of origin. The lines obtained from nude mice inoculated with polyclonal LCL seem to have a restricted clonal representation, but were not monoclonal, as evidenced by analyses of their pattern of immunoglobulin synthesis.


Vascular basophilia in ocular and orbital tumors. The occurrence of vascular basophilia in ocular tumors has been a selective histologic feature of retinoblastomas. We recently observed a metastatic oat-cell carcinoma to the choroid which also demonstrated such a vascular hematoxyphilia. Histologic review of a variety of ocular and orbital metastatic carcinomas failed to yield a similar basophilic pattern. Examination of 100 consecutive retinoblastomas for vascular basophilia revealed an incidence of 6.0%. Similar material was not seen in any of 125 melanomas, including 10 with areas of necrosis. Histochemical studies showed the basophilic material to be DNA, and electron microscopy revealed the nuclear debris of pyknotic tumor cells to be continuous with identical material surrounding the adjacent blood vessels. The pathogenesis of vascular deposition of DNA in these two ocular tumors remains unclear. This finding most likely represents a form of tumor activity requiring comparatively healthy blood vessels to adequately precipitate liberated nucleic acids being filtered from the necrotic and degenerating tumor tissue.


Adenovirus types 8 and 19 infection of rabbit corneal organ cultures. Adenovirus types 8 and 19 are responsible for a large number of cases of acute follicular conjunctivitis, mainly in the form of epidemic keratoconjunctivitis (EKC). This study successfully demonstrates the establishment of adenovirus infection in rabbit corneal organ cultures. Normal rabbit corneas were maintained in culture without significant morphologic alterations for up to 7 weeks. Adenovirus types 8 and 19 replication in rabbit corneas was demonstrated by viral growth curves and subpassage studies. Intracellular localization of adenoviral antigens was confirmed with immunofluorescent techniques. The characteristic cytopathic effects of adenoviral infection were observed with the use of various histochemical stains. Thus an in vitro corneal organ culture model was established for the study of the pathogenesis of corneal lesions in adenovirus EKC.


Scanning immunoelectron microscopy markers. Ultrastructural analysis of the cell surface and of membrane components such as antigens or receptor sites by scanning immunoelectron microscopy (immuno-SEM) has been the subject of extensive investigation during the past few years. We review the various immunologic and cytochemical techniques applied to SEM, which have employed latex particles, viruses, bacteriophages, protein A, ferritin, gold granules, hemocyanin and peroxidase as markers, and the advantages and disadvantages of these techniques. From current data, it is clear that immuno-SEM has much to offer in determining the distribution of specific cell surface sites and in positive and unambiguous identification of cell types in heterogeneous cell populations.


Tantalum oxide, silica and latex: effects on alveolar macrophage viability and lysozyme release. Tantalum an experimental bronchographic material, may be retained in the lungs for a prolonged period following bronchography. The alveolar macrophage (AM) is a cell with potential for clearing tantalum particles from the airways. We studied the in vitro effects of tantalum oxide and two other particles, silca and latex, on rabbit AM viability and lysozyme release over 30 hours. Results indicate: 1) tantalum oxide, silica, and latex particles are ingested by rabbit AM in culture; 2) tantalum oxide and silica are both toxic to AM in vitro; and 3) tantalum oxide exerts its toxic effects less rapidly on AM than does silica. On the basis of these in vitro culture results we conclude that tantalum oxide may be toxic to alveolar macrophages in vivo. Delayed lung clearance of tantalum oxide particles may be due in part to their toxic effects on alveolar macrophages.


[Papillomatous lactic-duct adenoma (pseudoPaget's disease of the mamilla)]. Papillomatosis and adenomatosis of the nipple ducts can be interpreted clinically as Paget's disease. Three patients with subareolar papillomatosis and adenomatosis are described. The name papillary adenoma of the nipple ducts (pseudo-Paget's disease has been proposed. The differentiation between the papillary adenoma of the nipple ducts and the genuine Paget's disease needs histological examination. The differential diagnosis is very important, because local excision of the diseased ducts represents the treatment of choice of the papillary adenoma of the nipple ducts. Radical mastectomy is always necessary in genuine Paget's disease since the adjacent lactiferous ducts are involved by the carcinoma.


Application of direct cannulation and injection lymphangiography to the study of the canine cardiac and pulmonary efferent mediastinal lymphatics. Lymphangiograms of canine cardiac and pulmonary efferent mediastinal lymphatics were made by cannulation and injection of Ethiodol. Injections were made singly and serially. The mediastinal lymphatics and lymph nodes, which constitute the pathways of drainage of the heart and lungs, were delineated from the point of cannulation to the right and left inferior cervical region where the right lymphatic duct and thoracic duct are located. Lymphangiography reveals that the lymphatics which drain the heart and lungs may join to form common mediastinal lymphatic channels. Interconnections between mediastinal channels were demonstrated. The lymphatics terminated in the region of both the right lymphatic duct and thoracic duct in every subject. The so-called "cardiac node of Drinker" is usually a group of pretracheal nodes rather than a single node. The pretracheal nodes and those more cephalad receive drainage of lymph from both the heart and lungs. These studies suggest that lymph collected by cannulation of a "cardiac" lymphatic adjacent to the "cardiac node" will contain pulmonary as well as cardiac lymph. Thus the high flows reported by many investigators for "cardiac" lymph probably indicates that pulmonary lymph is mixed with cardiac lymph, and that the experimental data should be interpreted with this in mind.


Isoproterenol in comparison to WR-2721 as a chemoradioprotector of the rat parotid gland. A comparison was made of the radioprotective abilities of the chemoradioprotector WR-2721, an amino-thiol, and the beta-sympathomimetic secretogogue isoproterenol on the rat parotid gland. Using the dose-response curve of WR-2721 for gland weights as a basis for comparison, isoproterenol was found to offer significant and equal protection during both the acute (DMF, 2.5) and the chronic (DMF, 2.3) periods.


Attachment of acetylcholinesterase to structures of the motor endplate. The kinetics of AChE solubilization from intact motor endplates of mouse diaphragm, by collagenase, papain and hyaluronidase, was studied in parallel with the ultrastructural localization of AChE in treated neuromuscular junctions. Hyaluronidase did not solubilize more AChE from isolated motor endplate regions than Ringer's solution itself. Residual AChE activity could be demonstrated histochemically in motor endplates even after the plateau of solubilization by collagenase or papain was reached. Less than 35% of junctional AChE is left after collagenase, and less than 20% after papain treatment, as estimated by the percentage of AChE activity left in the isolated endplate region of the diaphragm after protease treatment. Cytochemically, both proteases had a similar effect on postsynaptic AChE. Residual AChE activity was distributed randomly, adhering to the sarcolemma of junctional clefts. Presynaptic AChE localized in the gap between axon terminal and Schwann cell appears to be resistant to collagenase but not to papain treatment. The mode of AChE attachment or the composition of the intercellular material in this gap may differ from that of the primary and secondary clefts.


Haemophilus influenzae periplasmic protein which binds deoxyribonucleic acid: properties and possible participation in genetic transformation. A protein which binds to either single-stranded or double-stranded deoxyribonucleic acid (DNA) but not to ribonucleic acid has been isolated by osmotic shock treatment of growing cells. This periplasmic protein differs from the principal intracellular binding protein in its greater thermolability and by the absence of salt-induced cooperativity in its interaction with single-stranded DNA. Certain mutant strains of Haemophilus influenzae defective in the DNA suptake steps of genetic transformation were found to be deficient in periplasmic DNA-binding protein, suggesting that this protein participates in the uptake of DNA in transformation.


Overview of selected basic research in autism. Basic research in autism is reviewed. There is mounting indication, but as yet inconclusive evidence, of unique physiologic disturbances etiologically related to autism. Additionally there is indication that some of the physiologic disturbances found in autistic children are also present in children with other developmental disorders. Children called autistic probably represent a complex of clinically similar manifestations in a variety of different subgroups of children, each subgroup representing a basically different physiologic disturbance. However, the possibility remains that there is only one basic disturbance that in varying degrees affects many body systems and thus manifests in a variety of overlapping syndromes. Objective markers are needed so as to allow the demarcation of subgroups of autistic children for further study. Possible markers may be decreased duration of postrotatory nystagmus, auditory evoked response deviations, lymphocytic hyporesponsivity, increased blood platelet serotonin efflux, and/or the presence of urinary DMT or bufotenin.


Methyl ester of hyaluronate is unable to stimulate exolipase formation by Serratia marcescens. Hyaluronate stimulated the formation of exolipase by Serratia marcescens. This ability was abolished when all carboxyl groups of hyaluronate were methyl esterified. Additional studies suggested that the biological inactivity of esterified hyaluronate should be ascribed to the reduced conformational order of the molecules rather than to their electroneutrality.


Extrachromosomal psi+ determinant suppresses nonsense mutations in yeast. The extrachromosomal psi+ determinant in the yeast Saccharomyces cerevisiae enhanced the expression of Mendelian UAA suppressors by 6- to 10-fold. The psi+ determinant by itself is a weak UAA suppressor that caused the production of approximately 1% of the normal level of iso-1-cytochrome c in a strain containing the UAA mutation cycl-72.


Nicotinamide adenine dinucleotide metabolism in Candida albicans. The functional pathways of nicotinamide adenine dinucleotide (NAD) biosynthesis and their regulation were studied in the dimorphic fungus Candida albicans. The presence of a functional endogenous pathway of NAD biosynthesis from tryptophan was demonstrated. In addition, nicotinamide served as an efficient salvage precursor for NAD biosynthesis but nicotinate was not utilized. The pathway for nicotinamide utilization involved nicotinate and nicotinate nucleotides as intermediates, suggesting that the failure to utilize nicotinate involves a transport defect. The mechanisms that regulate NAD levels during exponential growth operated to maintain constant NAD levels when NAD biosynthesis occurred exclusively from endogenous or salvage pathways or from a combination of the two. The regulation also operated such that the salvage pathway was preferentially utilized.


Regulation of phosphoglycerate phosphomutase in developing forespores and dormant and germinated spores of Bacillus megaterium by the level of free manganous ions. The large depot of phosphoglyceric acid (PGA) which is accumulated within spores of Bacillus megaterium is greater than 99% 3-phosphoglyceric acid (3-PGA). The 3-PGA depot is stable in forespores and dormant spores, but is utilized rapidly during spore germination. When spores were germinated in KBr plus NaF, the PGA depot was not utilized, but 13% of the 3-PGA was converted to 2-PGA. These data suggest phosphoglycerate phosphomutase as the enzyme which is regulated to allow 3-PGA accumulation during sporulation. Young isolated forespores, in which 3-PGA was normally stable, utilized their 3-PGA rapidly when incubated with Mn2+ plus the divalent cation ionophore X-537A; Mn2+ or ionophore alone or Mg2+ or Ca2+ plus ionophore was without effect. Young forespores contained significant amounts of Mn2+. However, forespore Mn2+ exchanged slowly with exogenous Mn2+ and was removed poorly by toluene treatment. This suggests that much of the forespore Mn2+ is tightly bound to some forespore component. Since phosphoglycerate phosphomutase from B. megaterium has an absolute and specific requirement for Mn2+, these data suggest that the activity of this enzyme in vivo may be regulated to a large degree by the level of free Mn2+. Indeed, the activity of this enzyme in forespore or dormant spore extracts was stimulated greater than 25-fold by Mn2+, whereas comparable extracts from cells or germinated spores were stimulated only two- to fourfold.


Electric and flow birefringence properties of myosin in aqueous urea and sodium pyrophosphate. The electric dipole moment of rabbit skeletal myosin was estimated from the electric and flow birefringence properties. Myosin formed small polydisperse aggregates (0.2-1.1 microM in length) with an apparent electric dipole moment of 5,000-20,000 Debye in aqueous urea or sodium pyrophosphate at low ionic strength. Permanent dipole moment contributed substantially to the apparent dipole moment. An anti-parallel association of myosin was suggested from the dependence of the apparent dipole moment on myosin concentration. Some interactions between myosin and C-protein were detected in 1 M urea by flow birefringence and analytical ultracentrifugation studies. The apparent dipole moment of myosin aggregates was less dependent on myosin concentration in the presence of C-protein.


The use of soybean trypsin inhibitors as phosphorylatable substrates for a rat liver protein kinase. Phosphorylation by a cAMP-independent rat liver protein kinase of protein substrates containing the structural feature required by mammary gland casein kinase (-Ser-X-Glu/Asp) has been demonstrated. In particular, the Bowman-Birk Soybean trypsin inhibitor, which is characterized, like other legume protease inhibitors, by clusters of acidic residues near the C-terminal side of seryl residue(s), proved to be a good model substrate for the protein kinase. Its phosphorylation, involving the Ser 65 residue, is apparently hindered by the binding of trypsin, while it is stimulated by unfolding induced by reduction and subsequent carboxy-methylation.


Studies on regulatory functions of malic enzymes. IV. Effects of sulfhydryl group modification on the catalytic function of NAD-linked malic enzyme from Escherichia coli. Reactions catalyzed by NAD-linked malic enzyme from Escherichia coli were investigated. In addition to L-malate oxidative decarboxylase activity (Activity 1) and oxaloacetate decarboxylase activity (Activity 2), the enzyme exhibited oxaloacetate reductase activity (Activity 3) and pyruvate reductase activity (Activity 4). Optimum pH's for Activities 3 and 4 were 4.0 and 5.0, and their specific activities were 1.7 and 0.07, respectively. Upon reaction with N-ethylmaleimide (NEM), Activity 1 decreased following pseudo-first order kinetics. Activity 2 decreased in parallel with Activity 1, while Activities 3 and 4 were about ten-fold enhanced by NEM modification. Modification of one or two sulfhydryl groups per enzyme subunit caused an alteration of the activities. Tartronate, a substrate analog, NAD+, and Mn2+ protected the enzyme against the modification. The Km values for the substrates and coenzymes were not significantly affected by NEM modification. Similarly, other sulfhydryl reagents such as p-hydroxymercuribenzoate (PMB), 5,5'-dithiobis(2-nitrobenzoate) (DTNB), and iodoacetate inhibited the decarboxylase activities and activated the reductase activities to various extents. Modification of the enzyme with PMB or DTNB was reversed by the addition of a sulfhydryl compound such as dithiothreitol or 2-mercaptoethanol. Based on the above results, the mechanism of the alteration of enzyme activities by sulfhydryl group modification is discussed.


Construction of a multilayer planar membrane applicable to ion-transport measurement. Multilayer planar membranes applicable to ion-transport measurements were constructed from egg yolk lecithin, egg yolk lecithin-cholesterol mixture, and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine between two tightly stretched cellulose sheets. While most of the phospholipids in the membranes were found by a spin label technique to be uniformly oriented with their long hydrocarbon chains perpendicular to the surfaces of the cellulose sheets, a small fraction of phospholipids were isotropically oriented in multilayer membranes. The amount of phospholipids with isotropic orientations decreased with increasing content of cholesterol in membranes and became zero in membranes of egg yolk lecithin-cholesterol mixture (molar ratio of 1: 0.67). The degree of orientation, S, of uniformly oriented phospholipids in membranes was also increased by adding cholesterol to the membranes. The orientation of phospholipids in membranes was rather stable in distilled water and in aqueous calcium chloride (1, 10, 100 mM), while a marked disordering of oriented phospholipids was induced in a aqueous solutions containing thymol, isopropanol, or butanol beyond certain specific concentrations. The membranes can be used for measurements of calcium permeation. An appreciable barrier function to calcium permeation was detected with these multilayer planar membranes as compared with control experiments using only cellulose sheets as membranes. A preliminary investigation suggested that changes in the orientational structure of phospholipids in the multilayer planar membranes are correlated with permeability properties of the membranes.


Evidence for bactericidal activity of polymorphonuclear leukocytes without phagocytosis. The relationship between phagocytosis and bactericidal action of polymorphonuclear leukocytes was examined by comparing the functions of cytochalasin D-treated leukocytes with those of the control. Measurement of phagocytotic and bacterial DNA-degrading activities using Escherichia coli prelabeled with [3H]thymidine revealed that phagocytosis and bacterial DNA degradation were inhibited by treatment with cytochalasin D to about 50 and 10% of the control, respectively. Nevertheless, the bactericidal activity of the cytochalasin D-treated leukocytes was almost the same as that of the control leukocytes; almost all the bacteria were phagocytized by the latter leukocytes. Under the same experimental conditions, the production and release of superoxide anions and hydrogen peroxide, which are both known to be involved in the bactericidal action of the leukocytes, were markedly increased by cytochalasin D. Release of several lysosomal hydrolases was also increased markedly by cytochalasin D treatment, except for myeloperoxidase. However, lactate dehydrogenase, a typical cytosolic marker, was not released by the same treatment. Thus, it is unlikely that the increase in the release of the above-mentioned bactericidal factors was due to decomposition of the leukocytes. These results indicate that the site of bactericidal action of cytochalasin D-treated leukocytes is not necessarily intracellular but may be around the external surface of the cells.


A role of membranes in the activation of a new multifunctional protein kinase system. A new multifunctional protein kinase, which normally exists as an inactive form in the soluble fraction in mammalian tissues, attaches to membranes to exhibit full enzymatic activity. A low concentration of Ca2+ is absolutely necessary for this activation. This process is reversible. cAMP shows no effect. The active factors in membranes are phosphatidylinositol, phosphatidylserine, phosphatidic acid, diphosphatidylglycerol, and phosphatidylethanolamine in that order. Phosphatidylcholine and sphingomyelin are far less effective. Cytoplasmic as well as other membrane fractions from various tissues are active in supporting the enzymatic activity. A possible role of this Ca2+ and phospholipid-activated protein kinase system in transmembrane control is proposed.


Evidence for the existence of a novel enzyme system. myo-Inositol-1-phosphate dehydrogenase in Phaseolus aureus. A novel enzyme system, myo-inositol-1-phosphate dehydrogenase, has been isolated from germinating mung bean seeds. The dehydrogenation and cleavage of myo-inositol 1-phosphate by this enzyme leads to the synthesis of a pentose phosphate which appears to be ribulose 5-phosphate. The pH optimum of the enzyme is 8.6; NAD+ is required as coenzyme and no other nucleotides can replace NAD+. Mono- or divalent cations are not essential for the enzyme activity. Stoichiometry of the  reaction suggests that 2 mol of NAD+ are reduced per mol of ribulose-5-P generated.


Isolation and characterization of cultured mouse T-lymphoma cells deficient in uridine-cytidine kinase. Two clones were isolated from mutagenized mouse T-lymphoma cells (S49) which are over 90% deficient in uridine-cytidine kinase. The first clone, AU-200-1, was isolated in two steps by virtue of its resistance to 6-azauridine; whereas the second clone, FU3-70G, was isolated in three steps after exposure to three increasing concentrations of 5-fluorouracil. Extracts of both the AU-200-1 and the FU3-70G cell lines lacked over 90% of the capacity of those from wild type cells to phosphorylate either uridine or cytidine. Furthermore, the uptake of radioactive uridine and cytidine from the medium by intact AU-200-1 and FU3-70G cells was less than 5% of that found for intact wild type cells. By growth rate experiments, these uridine-cytidine kinase-deficient cell lines have altered sensitivities to the toxic pyrimidine analogs, 6-azauridine, 5-fluorouracil, and 5-fluorouridine and thus have been useful in elucidating the biochemical determinants involved in the metabolism of these compounds.


Active site phosphohistidine peptides from red cell bisphosphoglycerate synthase and yeast phosphoglycerate mutase. Bisphosphoglycerate synthase (glycerate-1,3-P2 yields glycerate-2,3-P2) and phosphoglycerate mutase (glycerate-3-P formed from glycerate-2-P) are both phosphorylated by substrates at a histidine residue forming covalent intermediates which have been shown to function in the phosphoryl transfer reactions catalyzed by these enzymes (Rose, Z. B., and Dube, S. (1976) J. Biol. Chem. 251, 4817--4822). We have phosphorylated bisphosphoglycerate synthase from horse red blood cells with [U-32P]glycerate-2,3-P2, digested with trypsin, and purified the phosphopeptide. The amino acid sequence of the phosphohistidine peptide has been determined to be: His-Gly-Gln-Gly-Ala-Trp-Asn-Lys. In like manner, a phosphohistidyl peptide has now been purified from yeast phosphoglycerate mutase, for which the amino acid sequence is known (Winn, S. I., Watson, H. C., Fothergill, L. A., and Harkins, R. N. (1977) Biochem. Soc. Trans. 5, 657-659). The amino acid composition of the phosphopeptide indicates that histidine-8 was phosphorylated. The sequence of this peptide is closely homologous with the active site peptide from bisphosphoglycerate synthase. In yeast phosphoglycerate mutase, the denatured phosphoenzyme hydrolyzes with a single rate constant of 2.02 X 10(-4) s-1 at pH 3, 45 degrees C. The relevance of these observations to the enzymatic mechanism is discussed.


Characteristics of chylomicron binding and lipid uptake by endothelial cells in culture. Bovine vascular endothelial cells bind chylomicrons via a high affinity membrane receptor site. Subsequent to binding, the chylomicron apoprotein was neither internalized nor degraded by either sparse or confluent (contact-inhibited) cells. However, the adsorption of chylomicrons was associated with interiorization of chylomicron cholesteryl ester and triglyceride and the hydrolysis of these lipids to free cholesterol and unesterified fatty acids by a lysosome-dependent pathway. This pathway was active in both subconfluent and contact-inhibited cells. The chylomicron free cholesterol so produced inhibited endogeneous cholesterol synthesis measured in terms of the incorporation of [1-14C]-acetate into sterol. An excess of high density lipoprotein was 2- to 3-fold more effective in reducing both binding of chylomicrons and interiorization of chylomicron lipid than was low density lipoprotein. Chylomicron binding was not "down-regulated" by preincubation of the cells with low density lipoprotein or chylomicrons. The results are discussed in the context of cholesterol sources for contact-inhibited endothelial cells which do not interiorize low density lipoprotein cholesterol.


The fat cell adenylate cyclase system. Characterization and manipulation of its bimodal regulation by GTP. GTP evoked both an activatory and an inhibitory response from adipocyte adenylate cyclase. This paper describes the persistence of the bimodal response under a variety of assay conditions. Additionally, manipulations are described which eliminate one or other of these actions. Treatment of adipocyte plasma membranes with cholera toxin A1 peptide and NAD+ abolishes the inhibitory phase of GTP action while preserving the activating phase. Treatment of the membranes with p-hydroxymercuriphenylsulfonic acid eliminates the activatory phase while maintaining the inhibitory processes mediated by GTP in adipocytes normally coexist and operate through different pathways since either phase can be abolished leaving the other intact. Adenosine and its purine-modified analogs inhibit fat cell adenylate cyclase in the GTP inhibitory phase (Londos, C., Cooper, D. M. F., Schlegel, W., and Rodbell, M. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 5362-5366). When this effect of GTP is abolished by either cholera toxin or Gpp(NH)p pretreatment, the inhibitory action of adenosine analogs is also lost. These data suggest a central role for GTP in mediating both activation and inhibition of adenylate cyclase by agents which act through cell surface receptors.


Dansylated thyrotropin as a probe of hormone-receptor interactions. A strongly fluorescent 5-dimethylamino-1-naphthalene sulfonate (dansyl) derivative of bovine thyrotropin has been prepared. The dye-conjugated hormone is bioactive and shares, essentially unchanged, the membrane binding and adenylate cyclase stimulatory activities of the native hormone. Binding of 125I-labeled dansyl-thyrotropin to thyroid plasma membranes is sensitive to inhibition by gangliosides and, as is the case for the binding of 125I-thyrotropin, galactosyl-N-acetylgalactosaminyl[N-acetylneuraminyl-N-acetylneuraminyl]-galactosylglucosylceramide (GDIb) is the most potent binding inhibitor. Gangliosides interact with dansyl-thyrotropin, causing a large increase of the quantum yield and a 5- to 10-nm blue shift of the emission maximum of the hormone-bound naphthalene chromophore; gangliosides cause no change in the fluorescent properties of the free dye. The fluorescence enhancement caused by gangliosides can be specifically reversed by unlabeled thyrotropin. The effect of gangliosides on dansyl-thyrotropin fluorescence is strongly salt-dependent; salts cannot, however, reverse the formation of the dansyl-thyrotropin.ganglioside complex once it has formed. The salt data suggest that the association of the ganglioside with dansyl-thyrotropin is dominated by electrostatic interactions, but that salt-independent, short range interactions, most likely hydrophobic, dominate the dissociation of the dansyl-thyrotropin-ganglioside adduct. Sucrose gradient centrifugation, ultracentrifugation, and fluorescence polarization data indicate that the gangliosides are micellar in nature under the conditions of these experiments. Acid titration of dansyl-thyrotropin causes a marked quenching of dansyl fluorescence which in part reflects dissociation of the hormone into its constituent alpha and beta subunits. In the presence of GDIb, but not N-acetylneuraminylgalactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosylceramide (GDIa), pH-dependent quenching and subunit dissociation are essentially eliminated. Circular dichroism results and fluorescence polarization studies support the interpretation that the ganglioside interaction causes a conformational change in the thyrotropin molecule. The acid titration data together with differences in the ability of gangliosides to influence the tyrosine fluorescence of the thyrotropin molecule indicate that different gangliosides induce different conformational perturbations in the thyrotropin molecule.


Role of phospholipids in the structure and function of the thyrotropin receptor. Phosphatidylinositol, phosphatidylserine, and phosphatidylethanolamine interact with 125I-thyrotropin and inhibit its binding to thyroid plasma membranes; phosphatidylcholine is not similarly effective. The interaction has been monitored by column chromatography on Sephadex G-100 which shows, for example, that 125I-labeled thyrotropin forms an adduct with phosphatidylinositol but not with phosphatidylcholine. Formation of the 125I-labeled thyrotropin-phosphatidylinositol adduct is dependent on the phosphatidylinositol concentration but can be reversed by both unlabeled thyrotropin and excess membranes. The efficacy of the phospholipid interaction and the phospholipid inhibition of thyrotropin binding to thyroid membranes is paralleled by changes in fluorescence and fluorescence polarization imposed on the 5-dimethylamino-1-naphthalene sulfonate (dansyl) derivative of thyrotropin. These changes are reversed by unlabeled thyrotropin but not by prolactin, placental lactogen, or growth hormone; similar changes are not observed when phospholipids are incubated with dansylated growth hormone, prolactin, and placental lactogen. Monovalent potassium, sodium, and lithium salts neither prevent nor reverse the formation of the phospholipid-dansyl-thyrotropin adduct; these results contrast with the effects of the same salts on the formation of ganglioside adducts with dansyl-thyrotropin. Despite their ability to interact witw 125I-thyrotropin in solution, neither phosphatidylinositol, phosphatidylserine, nor phosphatidylethanolamine, when incorporated in a liposome, binds the 125I-labeled ligand. These same phospholipids have no effect on ganglioside binding of 125I-labeled thyrotropin when gangliosides are incorporated in a liposome. These phospholipids do, however, modulate the expression of the glycoprotein component of the thyrotropin receptor when it is imbedded in a liposome. The phosphatidylinositol in this case serves as a negative modulator, both by decreasing the incorporation of the glycoprotein component of the receptor into the liposome and by inhibiting the binding activity of the glycoprotein component which is incorporated. Speculation is offered as to a possible role of the phospholipids in the message transmission process which would be consistent with current studies demonstrating a direct interaction of acidic phospholipids with thyrotropin. The effect of phospholipids on liposomes containing the glycoprotein component of the thyrotropin receptor raises the possibility that phospholipids and, in particular, phosphatidylinositol, may also play a role in regulating the insertion and expression of this receptor component in thyroid plasma membranes.


NADH binding to porcine mitochondrial malate dehydrogenase. The binding of NADH to porcine mitochondrial malate dehydrogenase in phosphate buffer at pH 7.5 has been studied by equilibrium and kinetic methods. Hyperbolic binding was obtained by fluorimetric titration of enzyme with NADH, in the presence or absence of hydroxymalonate. Identical results were obtained for titrations of NADH with enzyme in the presence or absence of hydroxymalonate, measured either by fluorescence emission intensity or by the product of intensity and anisotropy. The equilibrium constant for NADH dissociation was 3.8 +/- 0.2 micrometers, over a 23-fold range of enzyme concentration, and the value in the presence of saturating hydroxymalonate was 0.33 +/- 0.02 micrometer over a 10-fold range of enzyme concentration. The rate constant for NADH binding to the enzyme in the presence of hydroxymalonate was 3.6 X 10(7) M-1 s-1, while the value for dissociation from the ternary complex was 30 +/- 1 s-1. No limiting binding rate was obtained at pseudo-first order rate constants as high as 200 s-1, and the rate curve for dissociation was a single exponential for at least 98% of the amplitude. In addition to demonstrating that the binding sites are independent and indistinguishable, the absence of effects of enzyme concentration on the KD value indicates that NADH binds with equal affinity to monomeric and dimeric enzyme forms.


Nucleoside diphosphate kinase activity in purified cores of vesicular stomatitis virus. Purified cores of vesicular stomatitis virus contain an enzymatic activity that converts GDP, UDP, and CDP into their corresponding triphosphates using ATP as the phosphate donor. Thus, the virion-associated RNA polymerase can synthesize mRNA normally in vitro even when one of the ribonucleoside triphosphates is replaced by its corresponding diphosphate. RNA synthesis does not proceed if ATP is replaced by ADP. Similarly RNA synthesis is impaired if CDP and UDP are present in the same reaction. The role of the nucleoside diphosphate kinase (NDP kinase, EC 2.7.4.6) in vesicular stomatitis virus mRNA synthesis in vitro is discussed.


Partial characterization of a glycoprotein comprising the NH2-terminal region of mouse tumor cell pro-adrenocorticotropic hormone/endorphin. The glycoprotein accounting for most of the nonadrenocorticotropic hormone (ACTH), non-beta-lipotropin (beta LPH) region of mouse tumor cell pro-ACTH/endorphin was purified from tumor cell culture medium and shown to contain 1/2 cystine residues. Preparations of the 16,000-dalton fragment-related material (referred to as 16K fragment) were heterogeneous with respect to size and charge. Despite this heterogeneity, a partial amino acid sequence for the NH2-terminal region of the molecule was determined by automated Edman degradationof the 16K fragment labeled by reduction and alkylation with [3H]iodoacetic acid or labeled biosynthetically with [3H]tryptophan. The sequence of 1/2 cystine and tryptophan residues in the mouse tumor 16K fragment can be aligned with one region of the amino acid sequence predicted from the cDNA for a bovine precursor to ACTH/beta LPH (Nakanishi, S., Inoue, A., Kita, T., Nakamura, M., Chang, A.C.Y., Cohen, S.N., and Numa, S. (1979) Nature 278, 423--427).


Assembly of the mitochondrial membrane system. DNA sequence of subunit 2 of yeast cytochrome oxidase. A cytoplasmic "petitie" mutant of Saccharomyces cerevisiae (DS200/A1) has been isolated and determined to contain mitochondrial genetic markers in the oxi 1 locus. This locus has previously been reported to code for the structural gene of subunit 2 of cytochrome oxidase (Cabral et al. (1978) J. Biol. Chem. 243, 297-304). The segment of mitochondrial DNA retained in DS200/A1 has a repeat length of approximately 4500 base pairs and based on DNA sequencing contains a 756-nucleotide-long sequence that has been identified as the structural gene of subunit 2 of cytochrome oxidase. The presumptive gene sequence generates an amino acid sequence consistent with the reported molecular weight and composition of subunit 2 of yeast cytochrome oxidase. The correctness of the deduced amino acid sequence is further supported by its extensive homology to the primary structure of bovine cytochrome oxidase. The DNA segment of DS200/A1 has been located on the wild type mitochondrial DNA by comparative restriction mapping. The orientation of the COOH and NH2 termini and the direction of transcription of the gene have been determined.


Fibrosarcomata and malignant fibrous histiocytomata arising in relation to enchondromata. Six cases are reported in which a fibrosarcoma or malignant fibrous histiocytoma developed in relation to an enchondroma in a long bone. Four of the tumours were fibrosarcomata, and two were malignant fibrous histiocytomata. Five of the six cases were in the distal femur, which is a common site for old calcified enchondromata or "cartilage rests". The age of the patients was between fifty-six and eighty-six with a mean of seventy. Four were women, Five died less than one year after presentation. The fibrosarcomata and malignant fibrous histiocytomata do not appear to have arisen directly from the tumour cartilage but from the dense fibrous tissue surrounding necrotic areas in the enchondromata by a process analogous to that responsible for the development of fibrosarcomata in bone infarcts and chronic osteomyelitis. The possibility that some "dedifferentiated" chondrosarcomata are forms of collision tumour is discussed.


Adrenocorticotropin and beta-lipotropin in the hypothalamus. Localization in the same arcuate neurons by sequential immunocytochemical procedures. Adrenocorticotropin and beta-lipotropin (beta-LPH) have been localized by immunoperoxidase methods in nerve cells and fibers of the hypothalamus and brain stem of the ewe. 6-mum sections were immunostained first for either ACTH or beta-LPH. The reaction products and the antibody complexes were then eluted completely from the tissue, and the same section was immunostained for the second peptide. Absorption of the primary antisera with a variety of peptide fragments of ACTH and beta-LPH demonstrated, immunocytochemically as well as by radioimmunoassay, that the ACTH and beta-LPH antisera were directed to the COOH- and NH(2)-termini of the peptides, respectively. Neither antiserum recognized any portion of the heterologous peptide. In the sequential staining procedure on the same tissue section, preincubation of the antisera with the homologous peptide abolished the staining, whereas preincubation with the heterologous peptide did not affect it, regardless of the order followed. Every nerve cell in the arcuate nucleus that contained ACTH also contained beta-LPH, but beta-LPH cells appeared, probably falsely, to be twice as numerous as ACTH cells. beta-LPH-positive fibers in and beyond the hypothalamus were also more numerous and stained more intensively than ACTH fibers. The salient exception was fibers in the infundibular zona externa, where the opposite was true. Our observations establish that ACTH and beta-LPH are contained in the same nerve cells They stongly favor biosynthesis in brain, probably from a common precursor molecule, as has been demonstrated in the pituitary gland. The complexity of the cytologic distribution pattern described suggests that the two peptides are not processed in the same manner by the nerve cell.


Regulation of secretion of parathormone and secretory protein-I from separate intracellular pools by calcium, dibutyryl cyclic AMP, and (1)-isoproterenol. Dispersed porcine parathyroid cells were incubated at calcium concentrations between 0.5 and 3.0 mM in the presence of 3H- or 14C-amino acids to label newly synthesized parathormone. Up to four times more hormone was secreted at the lower calcium concentration but its specific radioactivity, from 30 to 50 times that of the intracellular pool, did not change. Dibutyrl cyclic AMP doubled immunoactive parathormone secretion at each calcium concentration, but there was no increase in secretion of radioactive hormone if labeled amino acids and secretagogue were added simultaneously. Similarly, when the intracellular pool of parathormone was prelabeled with 3H-amino acids and then the cells were incubated in 14C-amino acids and dibutyryl cyclic AMP, the entire increase in hormone secreted was derived from the prelabeled pool. (1)-isoproterenol increased intracellular cyclic AMP and acted on hormone secretion in a manner indistinguishable from dibutyryl cyclic AMP. In similar double-label experiments dibutyryl cyclic AMP preferentially enhanced secretion of secretory protein-I, a calcium-regulated protein of the parathyroid of unknown function. Calcium, alone, inhibited the intracellular level of cyclic AMP in a concentration-dependent fashion. These data are consistent with the existence in the parathyroid cell preparation of two hormone and secretory protein pools that may be individually recruitable--one consisting of most recently synthesized protein, the other consisting of older "storage" protein. The data do not allow one to decide whether the two pools coexist within individual cells or whether, instead, they exist in separate cells of the dispersed gland preparation.


Functional changes in human leukemic cell line HL-60. A model for myeloid differentiation. Polar solvents induce terminal differentiation in the human promyelocytic leukemia cell line HL-60. The present studies describe the functional changes that accompany the morphologic progression from promyelocytes to bands and poly-morphonuclear leukocytes (PMN) over 9 d of culture in 1.3 percent dimethylsulfoxide (DMSO). As the HL-60 cells mature, the rate of O(2-) production increase 18-fold, with a progressive shortening of the lag time required for activation. Hexosemonophosphate shunt activity rises concomitantly. Ingestin of paraffin oil droplets opsonized with complement or Ig increases 10-fold over 9 d in DMSO. Latex ingestion per cell by each morphologic type does not change significantly, but total latex ingestion by groups of cells increases with the rise in the proportion of mature cells with greater ingestion capacities. Degranulation, as measured by release of beta-glucuronidase, lysozyme, and peroxidase, reaches maximum after 3-6 d in DMSO, then declines. HL-60 cells contain no detectable lactoferrin, suggesting that their secondary granules are absent or defective. However, they kill staphylococci by day 6 in DMSO. Morphologically immature cells (days 1-3 in DMSO) are capable of O(2-) generation, hexosemonophosphate shunt activity, ingestion, degranulation, and bacterial killing. Maximal performance of each function by cells incubated in DMSO for longer periods of time is 50-100 percent that of normal PMN. DMSO- induced differentiation of HL-60 cells is a promising model for myeloid development.


Localization of Na pumps in the tracheal epithelium of the dog. Binding of [3H]ouabain by the dog's tracheal epithelium shows a nonspecific component depending linearly on ouabain concentration, and a specific saturable component with a Km of 10(-7) M. Control experiments showed that the tracer taken up was not trapped within the extracellular space nor bound to tissue collagen. Inhibition of the saturable uptake by high K, metabolic inhibition, low Na, and low temperature indicated that binding was to Na/K ATPase. One-sided exposures of tissue sheets to tracer showed that the submucosal side took up 10 X as much tracer as the luminal. Autoradiography localized tracer uptake under all conditions to the cells' basolateral membranes.


Quantitative biochemical analysis of microtubule content in normal and transformed 3T3 cells. Microtubules in normal and transformed BALB 3T3 cells were preserved in a stabilizing medium and measured by a [3H]colchicine-binding tubulin assay, and compared to total cellular tubulin measured under nonstabilizing conditions. Essentially no change in tubulin or microtubule content was seen with changes in cell density or with changes in cellular morphology at various stages of growth of normal or transformed cells or induced by dibutyryl cAMP treatment of transformed cells. Of five cell lines transformed by a variety of agents, four had a significantly higher total tubulin content than untransformed 3T3 cells and all of them had an increased microtubule content. None of the transformed lines had a lower fraction of tubulin recoverable as sedimentable microtubules compared to untransformed cells, and in three of them this fraction was significantly higher. These results establish that microtubules are present in transformed cells to at least the extent (if not greater) than in normal cells but that there are variations in the total amount of tubulin and microtubules as well as the fraction of the total tubulin present as microtubules which are not strictly correlated with transformation or cell morphology.


Dibutyryl cyclic AMP suppresses mobility of embryonic chick heart cells in aggregates. The intermixing of chick embryonic heart ventricle cells was studied in cellular aggregates as a function of embryonic age of the cells. Cell mobility, as measured by intermixing of radiolabelled and unlabelled cells, was high in cells derived from young embryos: 5.36 +/- 1.05 cell diameters per 2.5 days for 6-day-old heart ventricle cells. During development, mobility steadily declined to 1.56 +/- 0.24 cell diameters per 2.5 days for 18-day old cells. Treatment of aggregates with 1.0 mM theophylline plus 1.2 mM dibutyryl cyclic AMP resulted in greatly decreased mobility, particularly in aggregates of the more mobile younger cells. Depending on the embryonic age of the heart ventricle cells, this treatment reduced mobility by 45.3 to 89.4%. These data are consistent with an age-dependent decrease in intrinsic mobility superimposed upon a contact-paralysis mediated inhibition of movement present in solid tissues. In addition, the sensitivity of heart ventricle cells to inhibition by agents that increase intracellular cyclic AMP levels suggest that this is another possible inhibitory mechanism, although its physiological significance has not been established.


Identification of human anterior pituitary cells by immunoelectron microscopy. An attempt was made to classify human pituitary cell types by electron microscopic immunohistochemistry. The immunoperoxidase technique involving the use of the peroxidase-antiperoxidase complex was applied to thin sections of human pituitaries removed surgically for breast cancer or diabetic retinopathy. Using specific antibodies against human PRL, GH, beta-FSH, beta-LH, beta-TSH, and porcine ACTH, the localization of each hormone was studied. Identification of 5 human pituitary cells was possible: 1) The PRL-secreting cell contains round or slightly ovoid secretory granules of a diameter of 275-350 nm. 2) The GH-secreting cell is densely granulated with granule diameters ranging from 350-500 nm. 3) The gonadotrophic cell, which stains for both beta-FSH and beta-LH, is characterized by the presence of a varying number of secretory granules ranging from 275-375 nm. 4) The cortico-lipotrophic cell has numerous granules of about 375-550 nm in diameter. 5) The TSH-secreting cell contains small secretory granules of about 125-200 nm in diameter. Another cell type of which the small secretory granules of about 100 nm in diameter could not be stained by any of the antisera was also observed. This ultrastructural identification of human pituitary cells should contribute to a better understanding of the pathophysiology of the human pituitary.


Plasma aldosterone response to ACTH in primary aldosteronism and in patients with low renin hypertension. ACTH alpha 1-24 was infused at incremental rates of 12.5-200 mIU/30 min in dexamethasone-suppressed hypertensive patients on a regular sodium diet. The plasma aldosterone response to this stimulus in 8 patients with hyperaldosteronism due to an adrenal aldenoma and 11 with adrenal hyperplasia was significantly greater at all infusion rates (P less than 0.05) when compared with the response in 6 normal subjects on a similar diet. This responsiveness to ACTH in the patients with primary hyperaldosteronism was similar to that of the normal subjects on a low sodium diet. Twelve patients with low renin and 6 patients with normal renin essential hypertension were similarly studied. There was no significant difference in the median aldosterone response between these 2 groups and the normal subjects on a normal diet, but the response was significantly lower compared with that in patients with primary hyperaldosteronism. These data show that patients with hyperaldosteronism from an adrenal adenoma or hyperplasia have a consistent and exaggerated response to ACTH. The hyper-responsiveness is not apparently shared by the majority of patients with low renin essential hypertension and does not support the concept that this group is an intermediate form of primary aldosteronism. Individual patients within this group, however, may have such a response and might be identified by this type of testing.


Thyroglobulin inhibition of thyrotropin binding to thyroid plasma membrane. The effect of thyroglobulin (TG) on binding of TSH to thyroid plasma membranes was studied in vitro. Human and bovine thyroid plasma membranes have specific binding sites for bovine [125I]TSH. The binding of [125I]TSH is inhibited by the addition of purified TG (100 ng/microgram/ml). The inhibitory mechanism appears to be noncompetitive when subjected to Lineweaver-Burk analysis. Incubation of TG with TSH did not show an interaction, as assessed by sucrose gradient centrifugation. Plasma membranes prepared from human thyroid tissue have specific binding sites for human TG, as shown by [125I]TG binding assay. The TG binding was not affected by adding low concentrations of unlabeled bovine TSH. In the presence of very high concentrations of TSH, TG binding was increased. Hemoglobin, beta-lactoglobin, and ovalbumin did not have an inhibitory effect on [125I]TSH and [125I] TG binding to membrane preparations. Both [125I]TSH and [125I]TG binding were inhibited by 10 mM neuraminic acid. These results suggested that 1) TG released from thyroid gland may have a regulatory effect on TSH binding to its specific receptor, and 2) there are specific binding sites for TG on the thyroid plasma membrane.


Inhibition of leukocyte superoxide anion production by cortisol administration to normal subjects. Superoxide anion production was suppressed in leukocytes obtained at two or four hours following oral administration of 50 to 100 mg of cortisol to normal subjects. The suppression lasted at least 4 hours but was not present at 24 hours. The measurement of superoxide anion production by leukocytes appears to be a convenient method for the serial estimation of an important biologic effect of corticosteroids in human subjects.


Characterization of lipotropin-, corticotropin-, and beta-endorphin-immunoreactive materials secreted in vitro by a human pituitary adenoma responsible for a case of Nelson's syndrome. A human pituitary adenoma responsible for a case of Nelson's syndrome was maintained in organ culture and the incubation medium was examined with four different RIAs; human corticotropin (ACTH), beta-MSH, lipotropin (LPH), and beta-endorphin (beta-End). All four immunoreactivites (IRs) were present in the medium obtained after 24 h of incubation. Gel exclusion chromatography under  denaturing conditions (6 m guanidine HCl) revealed several immunoreactive components. Two components having both human beta-MSH (beta-hMSH) and human LPH (hLPH) IR coeluted with beta-hLPH and gamma-hLPH; a component with beta-hMSH IR but no hLPH IR coeluted with [125I]beta-hMSH; a component with human ACTH (hACTH) IR eluted at the position of hACTH. Sephadex G-50 gel exclusion chromatography revealed that approximately 80% and 20% of human beta-End (beta-hEnd) IR were accounted for by components coeluting with beta-hLPH and beta-hEnd, respectively. These data demonstrate the presence in this incubation medium of materials similar to if not identical with beta-hLPH, gamma-hLPH, hACTH, beta-hMSH, and beta-hEnd; they suggest that all of these peptides may be secreted in the circulation of patients with Nelson's syndrome.


Roentgenographic contrast agents inhibit triiodothyronine binding to nuclear receptors in vitro. The ability of roentgenographic contrast agents to inhibit binding of [125I]T3 to nuclear receptors was studied during incubation of rat liver nuclei or nuclear extracts in vitro and after ip administration of the agents in vivo. Ipodate, iodipamide, iopanoic acid, and diatrizoate inhibited binding of [125I]T3 in vitro. The most potent inhibitor was ipodate, which produced 50% inhibition of binding at 1.2 X 10(-4) M. When given orally in acute in vivo experiments, ipodate did not diminish binding to liver nuclear receptors. Ipodate appeared to inhibit in vivo metabolism of [125I]T3.


Effect of inhibition of prostaglandin synthesis on sympathetic nervous system function in man. The effect of inhibition of prostaglandin synthesis by indomethacin on the function of the peripheral sympathetic nervous system was studied in eight normotensive subjects. Sympathetic nervous function was assessed by measurement of plasma norepinephrine, alpha-adrenergic receptor sites on platelet membranes, and urinary excretion of epinephrine and norepinephrine. Treatment with indomethacin for 7 days resulted in significant decreases in basal plasma norepinephrine from 134 +/- 7 to 99 +/- 6 (SEM) pg/ml (P less than 0.01), a 26% decrease. Posturally stimulated norepinephrine concentrations (337 +/- 14 pg/ml in control studies) were 255 +/- 18 pg/ml (P less than 0.02), 25% lower, with indomethacin. Plasma norepinephrine after 5-min compression of hand grip (468 +/- 47 pg/ml in control) was 331 +/- 30 pg/ml (P less than 0.005), 29% lower, with indomethacin. The number of platelet alpha-adrenergic receptor sites did not change with indomethacin, nor did prostaglandin E1-stimulated cAMP production by platelet membranes. In addition, indomethacin produced no change in urinary excretion of norepinephrine or epinephrine. It is suggested that inhibition of prostaglandin synthesis may lead, via baroreceptor feedback, to a decrease in plasma norepinephrine concentration.


Dissociation of cortisol and adrenal androgen secretion in patients with secondary adrenal insufficiency. Plasma cortisol, dehydroepiandrosterone (DHA), dehydroepiandrosterone sulfate (DHAS), and androstenedione (delta4-A) were measured by RIA during ACTH infusion in preadrenarchal children with constitutional short stature, normal adults, and patients with secondary adrenal insufficiency resulting from hypothalamic-pituitary disease or corticosteroid therapy. The plasma levels of all four steroids were decreased in patients with secondary adrenal insufficiency compared to normal adults, but the decrease in DHA and DHAS was considerably greater than that in cortisol and delta4-A, resulting in significant decreases in the plasma ratios of DHA to cortisol, DHAS to cortisol, DHA to delta4-A, and DHAS to delta4-A (P less than 0.00001). The decreased DHA and DHAS responses to ACTH persisted in one glucocorticoid-treated patient after glucocorticoid therapy was terminated and the cortisol response to ACTH had normalized. The data suggest that adrenal atrophy due to hypothalamic-pituitary disease or corticosteroid therapy is associated with a greater impairment in the secretion of the delta5 adrenal androgens DHA and DHAS than in the secretion of cortisol and delta4-A, and that the capacity to secrete cortisol and delta4-A recovers more rapidly than the capacity to secrete the delta5 adrenal androgens when corticosteroid therapy is withdrawn.


FSH stimulation of 3H-glucosamine-incorporation into proteoglycans by porcine granulosa cells in vitro. Porcine granulosa cells from small follicles were incubated in the presence of 1 muCi/ml 3H-glucosamine and various hormones. Proteoglycans were digested with protease, and the mucopolysaccharides (MPS) were recovered. Most of the labeled MPS was found in secretory products. Maximum incorporation of 3H-glucosamine into MPS occurred after incubation with FSH for 8--16 h. Cell concentrations of 0.5--4.0 x 10(6)/ml proved to be ample. Highly purified porcine or human FSH gave consistent, significant stimulation by doses in the order of 100 pg/ml. Linear log-dose response curves occurred from 0.1 to 10.0 ng/ml, and maximal stimulation at 10 ng/ml was 22-fold above control. Preparations of hCG, hTSH and NIH-LH-S18 were not effective. Incubations with 1--10 mM dibutyryl cyclic AMP significantly increased incorporation into MPS, suggesting that effects of FSH may be mediated by cyclic AMP.


Isolation of oxidase-negative Pseudomonas aeruginosa from sputum culture. Two isolates of Pseudomonas aeruginosa lacking characteristic indophenol oxidase were recovered from a sputum specimen. A discussion of the characteristic biochemical tests and antibiograms along with a possible explanation for this phenomenon is presented.


Functional profile of the isolated uremic nephron: potassium adaptation in the rabbit cortical collecting tubule. As a renal function declines in patients and experimental animals with chronic renal disease, potassium homeostasis is maintained by a progressive increase in potassium secretion by the surviving nephrons, a phenomenon known as potassium adaptation. To determine the nephron site and the underlying mechanisms responsible for this phenomenon, studies were performed on normal and 75% nephrectomized rabbits maintained on normal or high-potassium diets. Cortical collecting tubules (CCT) were dissected from the normal and remnant kidneys and perfused in vitro in an artificial solution. In normal CCT mean (+/- SE) net K secretion, JK, (peq/cm per s) was 1.26 +/- 0.43 (normal diet) and 3.27 +/- 0.66 (high-K diet). In uremic CCT, JK was 3.55 +/- 0.60 (normal diet) and 6.83 +/- 0.58 (high-K diet). By reducing the dietary intake of potassium in proportion to the reduction of renal mass in these uremic animals, the adaptation in K secretion was prevented (JK: 1.22 +/- 0.40). Transepithelial potential difference was similar in CCT from normal and uremic animals on a normal diet despite the fact that JK was significantly greater in the latter group. However, in both normal and uremic CCT, the increase in JK caused by potassium loading was associated with an increase in luminal negativity. Uremic CCT underwent significant compensatory hypertrophy regardless of the dietary intake or potassium secretory rates. Plasma aldosterone levels were elevated only in the uremic-high potassium rabbits suggesting that a mineralocorticoid effect on the CCT may be exaggerated when potassium loading is superimposed upon decreased excretory capacity. The activity of Na-K ATPase was comparable in normal and uremic CCT from rabbits on either normal or high-K diets indicating that potassium adaptation may occur independently of changes in the activity of this enzyme. Intracellular potassium content measured chemically and by 42K exchange, was not significantly altered in either normal or uremic CCT when dietary potassium intake was increased, despite the fact the JK was increased under these circumstances. These data indicate that the CCT is an important site of potassium adaptation in the surviving nephrons of animals with reduced renal mass. This adaptation is an intrinsic property of the CCT and is expressed in the absence of a uremic milieu. Potassium adaptation by the uremic CCT is not fixed according to the degree of compensatory hypertrophy but varies according to the excretory requirements of the animal. Transepithelial potential difference and circulating aldosterone levels contribute to the adaptation but neither factor can entirely account for the phenomenon. Potassium adaptation by the CCT occurs in the absence of changes in Na-K ATPase activity and intracellular potassium content.


Regulation of primate testicular luteinizing hormone receptors and steroidogenesis. The testicular luteinizing hormone (LH) receptors of the rhesus monkey and human have many features in common, including high equilibrium association constant, marked species specificity, and relatively low binding capacity. We have, therefore, used rhesus monkeys as models for human LH-receptor regulation in vivo during gonadotropin treatment. In four adult male monkeys, treated with 10,000 IU human chorionic gonadotropin (hCG), serum and testicular steroidogenic responses were monitored at 24-h intervals during the following 4 d, and LH-receptor concentrations were measured by (125)I-hCG binding to 15,000-g particles prepared from testis biopsy specimens. In treated animals, serum hCG was maximal on day 1 at 322+/-16 ng/ml and declined to 24.4+/-2.3 ng/ml by day 4. Serum testosterone was increased threefold during the subsequent 4 d (from 6.5+/-2.0 to 18.6+/-4.4 ng/ml) but serum progesterone remained unchanged. In contrast, serum 17alpha-hydroxyprogesterone increased 12-fold to 5.5+/-0.5 ng/ml at day 1 and was increased fourfold during the subsequent 3 d. The LH-receptor binding capacity of the primate testis was reduced by 18.3+/-6.0% on day 1, 51.7+/-7.4% on day 2, and 45.3+/-2.4% on day 4. Occupancy of the LH receptors by endogenously bound hCG was significant on day 1 but was negligible by day 4. These data demonstrate that gonadotropin-induced LH-receptor depletion occurs in the rhesus testis and indicate that primate gonadotropin receptors are susceptible to the regulatory processes recently described in the rat. In addition, the simultaneous and disproportionate accumulation of 17alpha-hydroxyprogesterone indicates that 17,20-desmolase was rate-limiting under these conditions in the primate testis Leydig cell.


Phenobarbital-induced alterations in the metabolism of [3H]vitamin D3 by the perfused rachitic rat liver in vitro. Anticonvulsant therapy of seizure disorders in man is associated with the development of complications involving bone and mineral metabolism including hypocalcemia, elevated serum immunoreactive parathyroid hormone levels, and increased amounts of unmineralized bone or osteoid. The latter has been attributed to a reduction in serum-25-hydroxycholecalciferol levels resulting from increased hepatic metabolism of vitamin D. Using an in vitro recycling hepatic perfusion system, we have demonstrated that 5 d of phenobarbital treatment increases the hepatic production of [(3)H]25-hydroxyvitamin D(3) (4.3+/-0.3 vs. 3.3+/-0.2%/h, P <0.025) without affecting the biliary excretion of radioactivity. Furthermore, rachitic livers perfused with blood obtained from animals treated with phenobarbital for 5 d also manifested an increase in [(3)H]25-hydroxyvitamin D(3) production (4.6+/-0.5 vs. 3.3+/-0.2%/h, P < 0.02). Addition of phenobarbital or its major metabolite, p-hydroxyphenobarbital, directly to the perfusion apparatus had no effect on [(3)H]25-hydroxyvitamin D(3) production. Phenobarbital treatment was also attended by a decrease in the intrahepatic content of [(3)H]vitamin D(3) (11.7+/-0.4 vs. 17.5+/-0.7 dpm/mg liver protein, P < 0.001) without alterations in the content of [(3)H]25-hydroxyvitamin D(3). The data collectively suggest that the increased hepatic conversion of [(3)H]vitamin D(3) to [(3)H]25-hydroxyvitamin D(3) attending phenobarbital treatment is secondary to stimulation of the hepatic 25-hydroxylation system(s) by a metabolite of phenobarbital other than p-hydroxyphenobarbital and/or by metabolic alterations resulting from phenobarbital therapy.


Chemotactic factor inactivation by the myeloperoxidase-hydrogen peroxide-halide system. Polymorphonuclear leukocytes may modulate the acute inflammatory response by the secretion of enzymes capable of inactivating mediators of inflammation. The ability of the myeloperoxidase-H(2)O(2)-halide system of the neutrophil to inactivate chemoattractants was examined using both a radioassay and a morphologic assay of chemotaxis. Incubation of either a complement-derived agent, C5a, or a synthetic formyl-methionyl peptide chemoattractant with the myeloperoxidase system for 15 min at 37 degrees C resulted in essentially complete loss of chemotactic activity. Inactivation was dependent on enzymatically active myeloperoxidase, H(2)O(2) or a peroxide-generating enzyme system, and a halide cofactor. It was blocked by agents which inhibit peroxidase (azide) or degrade H(2)O(2) (catalase). Inactivation of chemoattractants was time-dependent, reaching maximal levels within 1-5 min, and temperature-dependent with no significant inactivation occurring at 0 degrees C. H(2)O(2) alone had no significant inactivating ability at concentrations as high as 10 mM, whereas in the presence of myeloperoxidase and a halide, 0.1 muM H(2)O(2) showed significant activity and 10 muM H(2)O(2) caused complete inactivation. On a molar basis, the order of effectiveness of the halide cofactors was Br(-) > I(-) > Cl(-), although only chloride was fully active at physiologic concentrations. Neutrophils stimulated by phagocytosis or by membraneperturbing agents secrete enzymatic constituents, including myeloperoxidase, and metabolic products such as H(2)O(2). Thus, it is suggested that the myeloperoxidase system acting at an extracellular site serves as an inflammatory control mechanism by virtue of its ability to inactivate neutrophil chemoattractants.


Metabolic fate of chylomicron phospholipids and apoproteins in the rat. To study the metabolic fate of chylomicron phospholipid and apoproteins, 15 mg of doubly labeled ([(3)H]leu, [(32)P]phospholipid) rat mesenteric lymph chylomicrons were injected as an intravenous bolus into conscious rats. The specific radioactivity, composition, pool size, and morphology of the plasma lipoproteins were determined after 2-60 min. After injection of chylomicrons, there was a rapid transfer of radioactivity into high density lipoproteins (HDL). At peak specific activity in HDL (2-5 min), 35% of injected apoprotein and 25% of phospholipid radioactivity were recovered in HDL (d 1.063-1.21 g/ml), with smaller recoveries in other lipoproteins and liver. There was an initial rapid rise of (32)P specific activity in HDL and d 1.02-1.063 lipoproteins (low density lipoproteins [LDL]), but whereas LDL specific activity subsequently converged with that of d < 1.02 lipoproteins, HDL specific activity decayed more rapidly than LDL or d < 1.02 lipoproteins. Lipolysis of chylomicrons was associated with a transfer of phospholipid mass into LDL and HDL. At 5 min, 80% of injected triglyceride had been lipolyzed and there was a significant increase in phospholipid mass in LDL and a smaller increase in HDL. At 10 min, the mass of phospholipid in LDL had returned towards control values, and there was a further increase in phospholipid mass in HDL, which suggested phospholipid transfer from LDL to HDL. In donor lymph chylomicrons (3)H-radioactivity was present in apoprotein (apo)B, apoA-I, and apoA-IV, but only radioactivity of apoA-I and apoA-IV were transferred to HDL. Transfer of radioactivity was associated with loss of mass of apoA-I and apoA-IV from the fraction that contained the chylomicron remnants (d < 1.02). With injection of 15 mg chylomicron, there was a small but insignificant increase in the relatively large pool of HDL apoA-I. However, 60 min after injection of 250 mg of human or rat intestinal chylomicrons into the rat, there was a significant increase in HDL apoA-I that resulted from acquisition of a major fraction of the chylomicron apoA-I. After injection of chylomicrons, phospholipid vesicles were observed by negative stain electron microscopy in the LDL and HDL ultracentrifugal fractions, especially in the LDL. Upon addition of an osmotically active compound, cellobiose, vesicles were observed as flattened particles with a double lipid bilayer thickness ( congruent with 100 A). To validate further the identity of these particles, chylomicrons were injected into rats with [(3)H]glucose, and the recipient rats' plasma was fractionated by chromatography on 6% agarose. Trapping of [(3)H]glucose occurred in the void and LDL regions of the column, and vesicular particles were identified in these column fractions by negative stain electron microscopy. Catabolism of chylomicrons is associated with a rapid transfer of phospholipid, apoA-I, and possibly apoA-IV into HDL. Chylomicron phospholipid appears to give rise to vesicles which are probably incorporated into preexisting HDL. Chylomicron surface components may be an important source of plasma HDL.


Maternal stress and pituitary-adrenal manipulations during pregnancy in rats: effects on morphology and sexual behavior of male offspring. The purpose of the present study was to determine whether the demasculinizing and feminizing effects of prenatal stress (i.e., stress applied to the mother during pregnancy) in rats reported previously are mediated by activation of the maternal pituitary-adrenal axis. Neither whole-body restraint, with or without hyperthermia, nor ACTH treatment during the last third of gestation had any reliable effect on masculine or feminine sexual behavior in male Sprague-Dawley offspring, although these treatments produced maternal pathology and evidence of maternal adrenocorticoid release. Significant littermate similarity was found for almost every morphological and behavioral measure. Failure to control for the litter variable may account for many previously reported effects of prenatal stress on sexual behavior in rats. The discrepancy between the present and earlier findings is discussed in terms of methodological and theoretical considerations.


Regulation of cyclic AMP metabolism and lipolysis in isolated rat fat cells by insulin, N6-(phenylisopropyl) adenosine and 2',5'-dideoxyadenosine. The increases in cyclic AMP accumulation and lipolysis by rat fat cells incubated in the presence of catecholamines were abolished by N6-(phenylisopropyl) adenosine. The same inhibition of cyclic AMP accumulation was seen in the presence of 2',5'-dideoxyadenosine but lipolysis was unaffected. In contrast, insulin inhibited lipolysis without affecting cyclic AMP accumulation by norepinephrine plus adenosine deaminase. These results suggest that there are either multiple pools of cyclic AMP or that ther exists some other mechanism which is involved in the regulation of lipolysis by hormones.


Comparison of cyclic nucleotide binding to adenosine 3',5'-monophosphate and guanosine 3',5'-monophosphate-dependent protein kinases. Binding sites for [3H]cAMP on purified regulatory dimers of type II A-kinase (II-R2) are independent as assessed by equilibrium binding (KD = 6 +/- 1.3 nM at pH 7.2, 25 degrees; nH = 1.0) and by the lack of effect of unlabeled cAMP on dissociation rate (kd = 1.0 X 10(-3) sec -1 at pH 7.2, 25 degrees). In contrast, binding sites for [3H]cGMP on purified G-kinase displayed positively cooperative interactions in both equilibrium and dissociation assays with convex upward Scatchard plots, an nH of 1.6 and a dissociation rate (kd = 6.2 X 10(-3) sec-1 at pH 6.8, 0 degree) which was slowed by excess unlabeled cGMP (kd = 1.13 X 10(-3) sec-1 at pH 6.8, degree). Calculated transition state free energies of dissociation revealed that dissociation of nucleotide from G-kinase in the presence of cGMP was restrained by an energy barrier (20.8 kcal.mol-1) similar to that of II-R2 (20.9 kcal.mol-1), whereas dissociation from G-kinase without excess nucleotide occurred more easily (18.9 kcal.mol-1).


Correlation between cyclic AMP levels and calcium efflux in isolated renal cortical tubules. Isolated renal cortical tubules from male hamsters were utilized to examine the possible relationship between cyclic AMP (cAMP) and efflux of calcium. Both parathyroid hormone (PTH) and prostaglandin E1 (PGE1) produced dose-related increases in cAMP levels and calcium efflux from isolated tubules. Maximal concentrations of both hormones resulted in changes in cAMP which were 6 fold greater and changes in calcium efflux which were 2 fold greater with PGE1 than with PTH. Effects of sub-maximal amounts of either hormone on both cAMP and calcium efflux were potentiated to tubule incubations resulted in increases in tissue-associated cAMP over the same degree by inclusion of methyl-isobutylxanthine (MIX). Addition of either exogenous cAMP or dibutyryl cAMP (db-cAMP) produced dose-related increases in calcium efflux which occurred more rapidly with db-cAMP than with cAMP. Increasing amounts of cAMP added to the same concentration range resulting in increases in calcium efflux. Addition of 2', 3' cyclic AMP, 5'AMP or db-cyclic GMP had no significant effect on calcium efflux while 3', 5' cyclic CMP significantly reduced this response. The results indicate that cAMP increases efflux of calcium from renal tubules and may play a central role in hormone-dependent transport of this ion.


Studies on the specificity of immunohistochemical techniques for cyclic AMP and cyclic GMP. Immunohistochemical studies employing antibodies against cyclic nucleotides indicate that cyclic AMP and cyclic GMP are localized to distinct subcellular sites. These antibodies, however, cross-react weakly with noncyclic nucleotides (eg. ATP, GTP), and therefore we investigated the speficity of the immunohistochemical technique. Slides of fetal nuclei exposed to gaseous nitrous acid demonstrated reduced immunofluorescence. The slides were then incubated with cyclic and noncyclic nucleotides, and restoration of distinct cyclic AMP and cyclic GMP staining pattern was achieved only with appropriate cyclic nucleotides. Antibodies that were used have a greater affinity for acetylated derivatives of cyclic nucleotides. By using a gas phase technique, tissue slices were acetylated and immunohistochemical staining intensity was compared with the effect of acetylation on antibody affinity for various nucleotides. Acetylation greatly increased affinity of cyclic AMP antibody for cyclic AMP but not other nucleotides, and greatly intensified cyclic AMP staining. Acetylation moderately increased affinity of cyclic GMP antibody for cyclic GMP, and moderately intensified cyclic GMP staining. Both nitrous acid and acetylation studies support the specificity of the immunohistochemical method for cyclic nucleotides.


Potential inhibitors of rat tooth alkaline phosphatase studied by means of different histochemical techniques. Two histochemical methods for demonstration of alkaline phosphatase activity, a lead pyrophosphate- anda naphtholphosphate technique, were compared. Since different results may be due to methodological differences as well as different enzyme activities, the enzymatic hydrolysis of the naphtholphosphate was visualized both by means of an azo-dye coupler and by lead-capturing of the liberated phosphate ion. Various potential inhibitors of alkaline phosphatase activity (diphosphonate, D-penicillamine, and sodium fluoride) were also tested. The use of diphosphonate and D-penicillamine resulted in inhibited or reduced staining, which could mainly be explained by an interference by these compounds with components in the incubation media rather than with the enzyme itself. The addition of sodium fluoride had no effect on the naphtholphosphate staining pattern irrespective of capturing method, whereas the odontoblastic pyrophosphate splitting alkaline phosphatase appeared to be sensitive to sodium fluoride, suggesting the presence of two alkaline phosphatases in odontoblasts.


Sleep apnea syndrome: report of case.

Sleep apnea syndrome: report of case. Sleep apnea syndrome encompasses several entities that are characterized by the inability of the victim to achieve restful sleep. The only known treatment for the syndrome is a tracheostomy. Further research and experimentation are required. We are of the opinion that the appliance described in this article offers some promise as an alternative treatment.


The demonstration of antibody specificity by a new technique. The gel electrophoresis-derived enzyme-linked immunosorbent assay (GEDELISA) and its application to antibodies specific for feline leukemia virus. By combining the high resolution of sodium dodecylsulfate polyacrylamide gel electrophoresis with the sensitivity of enzyme-linked immunosorbent assay (ELISA) antibodies specific for different feline leukemia virus components are characterized. Based on the same principle, Concanavalin A binding sites of FeLV components are also detected.


Ultrastructural demonstration of cytochrome oxidase via cytochrome C in cerebral cortex. A procedure for the ultrastructural cytochemical localization of cytochrome oxidase via cytochrome c in the cerebral cortex is described. Vascular perfusion fixation by formaldehyde and glutaraldehyde of different concentrations and mixtures of the two gave varying results. A mixture of 4% formaldehyde and 0.5% glutaraldehyde gave the best combination of ultrastructural preservation and retention of enzyme activity. Histochemical methods were examined for optimum incubation conditions, based on the oxidative polymerization of 3,3'-diaminobenzidine (DAB) to an osmiophilic product. The reaction product was discretely localized within intercristate and the intermembrane space of mitochondria. The staining pattern was the same in nerve cells and in neuroglia and their processed. The DAB reaction product was also found in mitochondria of the endothelial cells.


Morphogenesis of cerebral matrix ectopies in human fetus and newborn. The subject of investigations was genesis of periventricular, cerebral matrix ectopies in human fetuses and newborns. The morphologic and histochemical analysis concerned the embryonal elements of the ventricular wall: namely neuroepithelium with membrana limitans externa and suporficial layer of the germinal matrix. It has been distinguished four stages in the development of ectopic changes: 1) atrophy of neuroepithelium, 2) superficial necrobiosis with disruption of membrana limitans externa, 3) egsophytic neuroglial ectopies with organization or sequestration within the ventricular light, 4) neuroeptithelial inclusions in the periventricular zone. The disclosed, destructive changes in superficial layers of the germinal matrix as well as their glial organization suggested that reparative processes that could be an evidence of the passed perinatal hypoxia occur in the ventricular light.


Synaptic organization of the medial acessory olivary nucleus of the cat. Light microscopically the morphological characteristic of neurons and ultrastructurally the synaptic organization of medial accessory olivary nucleus of cat was studied. Different types of chemically mediated as well as electric synapses were observed. The terminal boutons contain mainly clear synaptic vesicles, but small and large dense core vesicles are also available. In the neuropil of the nucleus characteristic glomeruli with central dendritic core were establish. The dendritic profiles were connected with numerous gap junctions and attachment plaques. The apposition without any synaptic specializations in a large area of the cell membrane of adjacent neurons was also observed. In some cases oval structures with size 450--650 A which we called submembraneous dense bodies were established under one of the apposite membranes.


A prospective study of corticosteroid and catecholamine levels in relation to viral respiratory illness. Urinary 17-OHCS, epinephrine and norepinephrine levels were studied for one week before and one week after the onset of acute, severe Adenovirus 4 respiratory illness in 12 Army recruits during Basic Combat Training. During the pre-illness period, a tendency was frequently noted for all three hormone levels to show "spiking" elevations two to four days before illness onset. There was also a tendency for 17-OHCS levels to rise on the day before fever onset. The possible relationship of these pre-illness hormonal changes to stressful experiences and, in turn, to altered host resistance to infectious illness is discussed. Following onset of respiratory illness, 17-OHCS, epinephrine and norepinephrine levels all showed about 60 percent increases over the early pre-illness period baseline value. Elevations of these hormones persisted for about four to five days, roughly in correlation with fever duration, with only slight differences in configuration and timing of curves from one hormone to the next. The problem of evaluating which of several independent variables operating concurrently during infectious illness may be responsible for stimulating the final common neuroendocrine pathways is discussed.


Early events in thymocyte activation. II. Changes in nonhistone chromatin proteins induced by a thymus-dependent human serum factor. Previously, it has been shown that a human thymus-dependent serum factor (SF), isolated from peripheral blood and acting on precursors of mature T lymphocytes, induces an increase in the synthesis of cyclic AMP and proteins in thymocytes. We have now investigated the action of SF on the incorporation of 3H-leucine and 32P-orthophosphate into nuclear proteins of thymocytes after 15 to 240 min of culture. SF induced a rapid increase in the synthesis and phosphorylation of nuclear proteins, especially in the phosphorylated nonhistone chromatin proteins (P-NHCP). Electrophoretic patterns in polyacrylamide gels of the P-NHCP fractions, extracted from the chromatin of the stimulated cells, showed that proteins with m.w. higher than 50 x 10(3) were synthesized to a larger extent as compared with unstimulated cells. These data suggest that SF acts specifically on the synthesis of P-NHCP and may in this way control DNA-template activity.


Separation of functional subpopulations of murine and human lymphoid cells on colloidal silica density gradients. I. Preparation of the Ludox AM gradient material and characterization of separation capacities. This report describes a unique modification of an isopycnic density gradient system utilizing as a separating menstrum colloidal silica (Ludox AM). The primary advantages of this preparation are: (1) It is chemically defined, allowing extremely reproducible cell separation employing different lots of material; (2) the physical parameters (pH, density, salt concentration) of the final gradient suspension can be manipulated over a wide range of values, allowing for the separation of many different biological materials; (3) it allows separation of very large numbers of lymphoid cells with greater than 95% recovery of applied cells; (4) separated cellular subpopulations can be easily washed free of silica and cellular function is retained. This paper is a report of the preparation and functional characteristics of the gradient material as it relates to the separation of very large numbers of lymphoid cell subpopulations in both mouse and man. Subpopulations of murine and human lymphocytes separated by this gradient material were assayed for IgM synthesis, T-cell mediated cytotoxicity, and lymphokine production.


Surface analysis by bacterial adherence to virus-infected cells. A rapid and inexpensive assay has been developed for the examination of new antigens on the surface of whole eucaryotic cells. This assay is based on the specific tagging of antigen-antibody complexes at the cell surface with Staphylococcus aureus. Cells that had been infected with vesicular stomatitis virus were exposed to specific antiserum, were then exposed to the bacteria, and finally were examined in the light microscope. Almost 100% of infected cells reacted positively. Two temperature-sensitive mutants of vesicular stomatitis virus were employed to determine which virus-specific antigen interacted with antibody and resulted in adherence of bacteria to the cells. Examination of cells infected with either ts G41 or ts 045 at the nonpermissive temperature showed that the assay by bacterial adherence measured the presence of only the viral glycoprotein at the cell surface. Internal viral antigens or readsorbed extracellular virions did not result in bacterial adherence. This assay has potential widespread significance for diagnostic medicine as well as for rapid and sensitive analyses of cell surfaces.


A solid-phase radioimmunoassay for detection of IgM antibodies to hepatitis A virus. The conditions for a sensitive and specific solid-phase radioimmunoassay (RIA) for the detection of IgM antibodies to hepatitis A virus (HAV) were optimized, and the RIA was used to assay sera from patients with hepatitis. IgM antibodies to HAV reached highest concentrations between one and three weeks after onset of icterus and were measurable in follow-up sera for at least 12 months after infection. To prove the specificity, the IgG antibodies were separated from patient sera by sucrose density-gradient centrifugation. The remaining IgM antibodies, after treatment with beta-mercaptoethanol, did not bind in the RIA, and, when the anti-IgM antibody bound to the solid phase was replaced with anti-IgG, a negative result was obtained with incubation of IgM antibody to HAV. Also, the presence of IgG was shown not to interfere with measurement of IgM antibody to HAV. Finally, as a further specificity control, 50 sera positive for rheumatoid factor or from patients infected with hepatitis B virus, cytomegalic inclusion disease, infectious mononucleosis, influenza A virus, rubella, or measles were tested, and all of these sera were negative for IgM antibody to HAV.


Selective polymyositis inducted by coxsackievirus B1 in mice. A high-passage laboratory strain of coxsackievirus B1 produced a unique myositis that predominantly and profoundly affected hip extensors and, to a lesser extent, hindquarter knee flexors when inoculated into a strain of random-bred newborn mice of Swiss origin (COH mice). The effect was not observed in BALB/c or C3H mice similarly inoculated. In addition to the differences in susceptibility of the mouse strains, it was found that six different low-passage "field" isolates of coxsackie-virus B1 isolated from infected patients varied considerably in their capability to induce these lesions. Thus, selective myositis which is muscle group-specific can be induced in a mouse model with coxsackievirus B1, and both genetic factors of the host and characteristics of the virus strain play a significant role in the pathogenesis of the myositis.


Inhibition of the development of spontaneous hypertension in SH rats by gonadectomy or estradiol. Young male and female rats that become spontaneously hypertensive when they mature were gonadectomized at 30 days of age while they were still normotensive. Gonadectomy retarded the usual steep ascent of blood pressure up to 120 days of age. Ovariectomized females "escaped" from this inhibiting effect, and blood pressure rose to severely high levels, i.e., 210 mm Hg, from 150 to 240 days. Treatment with testosterone or estradiol demonstrated that estradiol was particularly effective in inhibiting the usual rise in blood pressure in intact (sham-operated) or gonadectomized males and females. Although effectively lowering blood pressure, estradiol also caused increased pituitary and adrenal weights, hyperlipidemia, and increased circulating levels of corticosterone and DOC.


Chemodectomas: review and report of nine cases. We have reviewed the broad spectrum of disease caused by chemodectomas. This spectrum extends from the benign to the aggressively malignant with many graduations in-between. Our analyses included cases from the literature and nine new cases seen over the past twenty-five years. Surgery as the primary and most definitive form of therapy, is recommended if feasible, with total excision as the goal, in both benign and malignant histologies. An excellent outcome is to be expected in benign cases. At present, no predictor exists to foretell the behavior of malignant lesions, which can range from the aggressive to the slowly progressive. Both radiotherapy and chemotherapy have been tried in malignant cases. No consistent good result has occurred from the use of either. The future will hopefully bring us more effective therapy.


Adrenocortical function and a field population of a macropodid marsupial (Setonix brachyurus, Quoy and Gaimard). Concentrations of corticosteroids and aldosterone have been measured in the plasma and related to the maximum cortisol binding capacity, to the concentration of sodium and potassium of the plasma and to the condition of the animals, in a natural population of the quokka (Setonix brachyurus, Quoy & Gaimard) on Rottnest Island, Western Australia. A marked population in one area has been repetitively sampled at approximately 6 week intervals over a period of 2 years. Animals were found to be in significantly better condition during the hot-dry season. Their plasma concentrations of corticosteroids, 1.09 +/- 0.11 (S.E.M.) and 1.47 +/- 0.14 microgram/100 ml and their plasma sodium concentrations, 147 +/- 0.87 and 142 +/- 1.17 mequiv/1, for the hot-dry and cold-wet seasons respectively, were both significantly different (P less than 0.05). Plasma concentrations of aldosterone were 8.40 +/- 0.57 and 7.06 +/- 0.53 ng/100 ml for the hot-dry and cold-wet seasons. The level of aldosterone fell to levels comparable to those found in salt-replete laboratory animals only during the month of July. In the laboratory, salt deprivation resulted in a significant rise in the concentration of plasma aldosterone, 2.33 +/- 0.57 (S.E.M.) ng/100 ml when salt-replete and 16.95 +/- 2.74 ng/100 ml when salt-deprived (P less than 0.001). Total plasma corticosteroid levels were unaffected by changes in the sodium content of the diet. Injections of ACTH at a rate of 2 i.u./kg caused a significant rise in the levels of plasma corticosteroids which were essentially identical in both high- and low-condition animals. Both temperature fell to a significantly lower level in low-condition animals held in cages after injection of ACTH. These results indicate that the seasonal mortality of th quokka on Rottnest Island does not result from any breakdown in adrenal function, despite a substantial decline in the condition of the animals.


Distribution and degradation of two tritium-labelled corticotrophin analogues in the rat. The distribution and degradation of corticotrophin-(1--24)-tetracosapeptide specifically labelled with tritium at Tyr2, Phe7 or Tyr23 and [D-Ser1, Lys17, Lys18]-corticotrophin-(1--18)-octadecapeptide amide labelled at Tyr2 were studied at various times after intravenous injection into rats. By characterizing the radioactivity in plasma and various tissues, an overall picture of the metabolic handling of the two peptides emerged. The peptides left the circulation rapidly, entering mainly muscle and skin where they were extensively degraded. The D-Ser1-containing analogue was less rapidly degraded and intact peptide persisted in muscle and skin for up to 1 h. This peptide probably returned to the circulation giving rise to the sustained plasma levels observed after injection of the D-Ser1-substituted octadecapeptide but not after injection of the tetracosapeptide. Although initially the kidneys did not clear such large amounts of peptide as did muscle and skin they played an important role by continuously and, on the basis of existing evidence, irreversibly clearing the peptides and peptide fragments from the circulation and degrading them.


Synaptic facilitation in Aplysia explored by random presynaptic stimulation. The identified interneuron L10 in the abdominal ganglion of Aplysia was stimulated to fire action potentials in a random sequence while the early inhibitory potential of its follower cell L2 was recorded. Application of Wiener nonlinear analysis to these data yielded a predictive model of the facilitating postsynaptic potential. The model shows that facilitation changes both the time-course and the magnitude of the early synaptic potential. The facilitated response has a longer duration than the unfacilitated response. Its magnitude is exponentially decreasing with increasing interstimulus interval between test and conditioning stimuli. Facilitation is abolished at short interstimulus intervals. The hypothesis that the magnitude only of transmitter release is increased cannot explain these results. The observed facilitation may be due to characteristics of pre- and postsynaptic morphology.


Light-initiated changes of cyclic guanosine monophosphate levels in the frog retina measured with quick-freezing techniques. Although there is good agreement that light reduces the amount of cyclic GMP (cGMP) in the retina, the exact time-course of this decrease is not well established. Bullfrog retinal sections were isolated under infrared light and quick-frozen with liquid nitrogen-cooled, metal hammers after exposure to various intensities of continuous illumination. This quick-freezing should stop the degradation of cGMP within 50-100 ms. The frozen retinal sections were then slowly warmed up in the presence of perchloric acid to denature enzymes involved in cGmp metabolism. cGMP was determined by radioimmunoassay and comparison was made between light- and dark-adapted retinal sections from the same animal. The average cGMP concentration was 44.3 +/- 0.7 pmol cGMP/mg protein or 170.9 +/- 3.2 pmol cGMP/retina. After 1 s of illumination no significant change in cGMP concentration was found even with the brightest light used (approximately 7 x 10(7) rhodopsins bleached/second per rod. At this intensity the first significant decrease in cGMP from dark-adapted levels was detected 3-5 s after the initiation of illumination; cGMP decayed to 70-75% of the dark-adapted value after approximately 30 s. With lower intensity illumination the cGMP levels recovered to dark-adapted levels after the initial decrease even though the bleaching light remained on.