The estrogen cytosol receptor of female ovine pituitary.

The biochemical parameters of estrone and estradiol binding to the cytosol fraction of ovine anterior pituitary were investigated. When increasing amounts of [3H]estrone or [3H]estradiol were incubated with the 105,000 g fraction from the pituitary, both hormones bound to a receptor with the same apparent KD (mean +/- S.E., estrone = 1.40 +/- 0.30 X 10(-10) M, estradiol = 1.03 +/- 0.11 X 10(-10)M) and the same concentration of binding sites (estrone = 3.22 +/- 0.58 X 10(-14) moles/mg protein, estradiol = 3.92 +/- 0.19 X 10(-14)). No conversion of [3H]estrone to [3H]estradiol under the experimental conditions used could be demonstrated. The receptor-estrogen complex exhibited identical sedimentation coefficients (7-8 S) with either hormone. The receptor was specific only for estrogens; neither 500-fold excess of testosterone nor progesterone affected binding. Competitive inhibition using increasing amounts of non-radioactive estrone or estradiol with [3H]estrone or [3H]estradiol resulted in parallel displacement of the radioactive hormone. These results strongly suggest that both hormones bind to the same pituitary cytosol receptor.


Hormonal regulation of glycogen metabolism in human fetal liver. I. Normal development and effects of dibutyryl cyclic AMP, glucagon, and insulin in liver explants.

Glycogen accumulates in human fetal liver beginning at the eighth week of gestation. A parallel increase in total glycogen synthase activity is found, although the I-form activity remains low and constant throughout the first two thirds of gestation. Total phosphorylase activity increases slightly during this period, with the proportion in the active form amounting to about one half of the total throughout. After an initial rapid decline, the glycogen concentration in explants of human fetal liver remained constant for twenty to forty hours at about 20 per cent of the in vivo level. Incubation with glucagon, cyclic AMP (adenosine 3',5'-monophosphate) or its dibutyryl derivative markedly reduced tissue glycogen concentrations while insulin brought about a small increase. The effect of maximal doses of dibutyryl cyclic AMP and glucagon were the same, and the combination of agents produced no further effect. The response to dibutyryl cyclic AMP was apparent by one hour and maximal by three to six hours, whereas the response to insulin required about six hours to be detected, and it continued for at least eighteen hours. Insulin antagonized the glycogenolytic effect of low doses of glucagon or theophylline but was without significant effect in the presence of high glucagon concentrations. Glucagon stimulated cyclic AMP output from explants, and this effect was further augmented by theophylline. Insultin had no consistent effect on cyclic AMP output in either the presence or the absence of glucagon or theophylline. Incubation with dibutyryl cyclic AMP resulted in a decrease of glycogen synthase I-form activity, while insulin tended to increase this enzyme activity. In neither circumstance was the proportion of active phosphorylase altered. These results suggest that the regulation of glycogen levels in human fetal liver by cyclic AMP, glucagon, and insulin may entail alterations in the activity of glycogen synthase activity without necessitating alterations in phosphorylase activity. Cyclic AMP or glucagon was capable of depleting tissue glycogen stores in tissue from fetuses of six weeks' gestation. Insulin increased tissue glycogen concentrations in tissue from fetuses of seven or more weeks.


Effect of Shigella enterotoxin on electrolyte transport in rabbit ileum.

Shigella dysenteriae I is one of several bacteria which produces an enterotoxin capable of stimulating intestinal water and electrolyte secretion. Unlike cholera and Escherichia coli enterotoxins which have been shown to increase intracellular cyclic adenosine monophosphate levels in the small intestine, the mechanism by which shigella enterotoxin causes intestinal secretion is not known. To study shigella enterotoxin-stimulated intestinal secretion, rabbit ileal mucosa exposed in vivo to Shigella dysenteriae I enterotoxin was studied in vitro in a modified Ussing chamber. Fluid and electrolyte accumulation occurred in vivo and net sodium secretion was present in vitro in the enterotoxin-exposed tissue in contrast to net sodium absorption in control mucosa. Short-circuit current (Isc) was similar in shigella enterotoxin-exposed tissue compared with control tissue. The increase adenosine monophosphate was similar to enterotoxin-exposed and control mucosa. The addition of glucose resulted in a smaller increment of Isc in shigella enterotoxin-treated tissue. Mucosal cyclic adenosine monophosphate levels in enterotoxin-exposed mucosa did not differ from those of control. These results indicate that the characteristics of rabbit ileal mucosa exposed to shigella enterotoxin and cholera enterotoxin markedly differ, although both produce electrolyte secretion both in vivo and in vitro. These studies further suggest that, in contrast to its role in cholera enterotoxin-induced intestinal secretion, cyclic adenosine monophosphate may not be in the mediator of shigella enterotoxin stimulation of intestinal fluid and electrolyte secretion.


Pathogenesis of Salmonella-mediated intestinal fluid secretion. Activation of adenylate cyclase and inhibition by indomethacin.

Salmonella typhimurium, an organism that invades intestinal mucosa but does not elaborate a traditional enterotoxin, evokes ileal secretion by causing alterations in active sodium and chloride transport mechanisms. To evaluate the possibility that these changes in transport might be related to the adenylate cyclase-cyclic AMP or NA+-K+-adenosine triphosphatase (ATPase) systems, mucosal adenylate cyclase, cAMP phosphodiesterase, Na+-K+ and Mg++ ATPase activities, and cAMP concentrations were measured in rabbit ileal loops infected with two strains of S. typhimurium. Strain TML invades the mucosa and evokes fluid secretion whereas strain SL 1027 invades but does not evoke secretion. Cholera toxin-stimulated loops were also studied. When compared to control loops, TML-infected mucosa demonstrated a marked increase in adenylate cyclase activity, in cAMP concentration, and no change in phosphodiesterase or ATPase activities. SL 1027-infected mucosa demonstrated no change in either adenylate cyclase or ATPase activities. Indomethacin pretreatment of cyclase activation. In contrast, indomethacin pretreatment of cholera toxin exposed animals resulted in only a partial reduction of secretion while not altering the stimulation of adenylate cyclase. These results suggest that: (1) S. typhimurium causes ileal secretion by stimulating adenylate cyclase; (2) mucosal invasion alone (SL 1027) is not sufficient to activate adenylate cyclase, and (3) Na+-K+-ATPase does not appear to be involved in salmonella-induced secretion. The mechanism of salmonella activation of adenylate cyclase is unclear but apparently differs from that of cholera toxin in that it is inhibited by indomethacin. This might be explained by the participation of prostaglandins in the salmonella activation process.


Etiological spectrum of post-transfusion hepatitis.

Frequent occurrence of post-transfusion hepatitis continues despite screening for Australia antigen in blood before transfusion and elimination of commercial donor sources. The majority of these cases appears unrelated to hepatitis B virus. Preoperative, acute, and convalescent serra were screened for evidence of hepatitis B, hepatitis A, Epstein-Barr, and cytomegalovirus exposure in 34 cardiac surgery patients developing post-transfusion hepatitis postoperatively. Four patients showed evidence of hepatitis B infection and 3 patients developed significant antibody titer rises to cytomegalovirus. No patient showed evidence for acute hepatitis A infection postoperatively in response to blood transfusions. Epstein-Barr virus was also not responsible for any cases of post-transfusion hepatitis. Presently available laboratory methods failed to implicate hepatitis A, Epstein-Barr, or cytomegalo-virus in the majority of non-B post-transfusion hepatitis cases. This suggests that identification and characterization of additional hepatitis-producing agents will be required to define further the epidemiology of post-transfusion hepatitis and develop measures for its prevention.


[Histological and histochemical studies on testis, epididymis and ductus defrens of the rooster (Gallus domesticus)].

Testis, epididymis and ductus deferens of the adult domestic fowl and male gonads of juvenile roosters have been studied by means of histochemical and histological methods. Testicular interstitial cells: According to enzyme-histochemical results oxidative energy production seems to be of minor importance. An extraordinarily high activity of lysosomal enzymes (acid phosphatase and esterases in the adult, esterases only in the immature) is observed. Positive reactions of 3beta-steroid-dehydrogenase and enzymes of the pentose phosphate cycle indicate steroid hormone production; the pathways of the steroid synthesis, however, are probably different in adult and immature testes. A remarkable LAP content of juvenile Leydig cells is a parameter of an increased protein metabolism. Areas of reserve cells: Focal accumulation of these cell types are observed in testis and epididymis of the immature and in the epididymis of the adult fowl. Reserve cells reveal distinct activities of LAP (prospective growth ability) and 3beta-HstDH (reserve capacity for steriod synthesis). All other enzymes studied react weakly, thus pointing to a generally low metabolic activity. Seminiferous tubules: The strong reaction of alkaline phosphatase in the peritubular cells may play a part in energy disposition for contractions. Sertoli cells of adult animals are rich in lysosomal enzymes and enzymes of the glycolytic chain but oxidoreductases react weaker than in mammalian Sertoli cells. This indicates that nutritive interactions between germ and Sertoli cells in birds are different from those in mammals: The basally orientated germ cells of birds contain strong activities of diaphorases, LDH, SDH, Cyto-Ox and seem to be metabolically rather indipendent from Sertoli cells.


Immune response and the B blood group locus in chickens.

Chickens of three blood group genotypes, B1B1, B1B2 and B1B19, were compared in their ability to produce antibodies. The B locus is the major histocompatibility locus in this species. Homozygous B1 pullets had significantly higher adult mortality than did the B1 heterozygotes. In two separate experiments the B1B1 females produced significantly fewer antibodies to Salmonella pullorum than did the B1 heterozygotes. Also the B1B1 pullets responded with lower antibody titers following immunization with ferritin, bovine serum albumin and parainfluenza-3 virus, although the differences were not significant. The results of this study suggest that an immune response gene is associated with the major histocompatibility locus in chickens, paralleling the H-2 locus and Ir genes in mice.


Platelet aggregometry and anti-platelet isoantibodies.

102 sera from polytransfused patients have been screened in platelet aggregometry. Anti-human platelet isoantibodies, tested against 'responsive' human platelets in PRP, give in the aggregometer a decrease in optical density recorded as a sigmoidal curve. In the first step of reaction PF3 availability suggests that the primary action of antibody is to damage the platelet membrane. The second step might be a true aggregation (an ADP-mediated platelet clumping) or a platelet lysis, depending on the type and the potency of antibody, on the type of platelets (normal or thrombasthenic) and on some experimental conditions. These conclusions, confirmed by electron microscopy findings, suggest that aggregometry is a very rapid and simple method of detecting platelet antibodies.


[Surgical treatment of congenital malformations of the hand].

The commoner and more important congenital deformities of the hand are reviewed, considering their clinical picture, indications and optimal age for intervention, operative technique, postoperative care, and results. It is stressed that the value of many of the procedures can be assessed only after several years, and often only at the end of the growing period. The following malformations are discussed: 1. Syndactyly: There are many patterns of deformity; fusion of fingers of unequal legnth and distal bony fusion with convergence are absolute indications for early separation (six months of age); in uncomplicated cases operation can wait until the second year of life. Correctly placed skin incisions are of great importance for the prevention of scar contractures; a zig-zag incision forms long triangular flaps for cover of the sides of the fingers, splitting up areas of full-thickness skin graft; the commissure is formed from either two triangular flaps or a single dorsal rectangular flap...


[Critical considerations of various surgical methods for correction of the syndactylia (report on an observation period of 10-20 years)].

During the postoperative period of 10 to 21 years 58 syndactylies in 45 hands of 30 patients were followed-up with special attention to alternations due to growth. These were prevented in the majority of the patients, if the skin cover for the commissure was done by both a dorsal and palmar triangular skin flap and for the side areas of the fingers by several transversely oriented full thickness skin grafts, separated by bridges of the finger skin. In none of the 37 syndactylies in which this procedure was performed, was a corrective operation necessary at the end of the growth period. These results harmonize with previous follow-up studies (MILLESI 1964). The observations of these patients for an additional 10 years have not shown any alterations due to growth.


Histochemical studies on the albino rat pancreas in different periods following vagotomy and simultaneous pyloromyotomy.

The effect of complete subphrenic vagotomy and simultaneous pyloromyotomy on the morphological state and the activities of some intracellular enzymes of the albino rat was studied histochemically. Within the first weeks after vagotomy, the pancreatic acini were found to diminish in size, and the beta-cells in the islets of Langerhans became oedematous. In the acini, the activities of succinate dehydrogenase, cytochrome oxidase, AS naphthol acetate esterase, and glucose-6-phosphatase were observed to decline, but the reactions for beta-glucuronidase and aryl sulphatase showed intensifications and polymorphic behaviour both in acinar and in islet cells. The latter also and particularly the beta-cells simultaneously revealed enhanced activities of succinate dehydrogenase, cytochrome oxidase, beta-glucuronidase, and aryl sulphatase, and an entire disappearance of the reaction for glucose-6-phosphatase. The alpha-cells increased their AS naphthol acetate esterase activity. After 5 weeks following vagotomy, morphological and enzymatic changes in the acini and islets were negligible, and after 5  and 9 months no differences were noted between the vagotomized rats and the control animals.


[Physiological role of prostaglandins in adrenergic and non-adrenergic inhibitory neurotransmission in guinea pig taenia coli].

Prostaglandin (PG) E1, PGE2 and PGF2alpha and their biosynthesis inhibitor, indomethacin, were tested for their effects on the inhibitory responses of taenia induced by electrical stimulation of adrenergic and/or non-adrenergic inhibitory nerves in the perivascular nerve-taenia preparation isolated from guinea-pig caecum. The response to adrenergic nerve stimulation was considerably reduced by PGE1 and PGE2, while it was little affected by PGF2alpha. Although PGE1 and PGE2 produced contraction of taenia, it may be possible to dismiss from consideration their action of contraction of taenia in accounting for their inhibitory effect on the response to nerve stimulation since the following observations were made, 1) when the taenia were contracted by histamine, the response to adrenergic nerve stimulation was not reduced and 2) in the presence of polyphloretin phosphate, PGE1 and PGE2 did not contract taenia but reduced the response to adrenergic nerve stimulation. In the presence of indomethacin, the response to adrenergic nerve stimulation was increased greatly. On the contrary, the inhibitory response to non-adrenergic inhibitory nerve stimulation was not affected by application of PGE1, PGE2, PGF2alpha and indomethacin. These results suggest that endogenous PG of E series in guinea-pig taenia may play a role in modulating adrenergic neurotransmission. Attempts to demonstrate that PG could operate on a non-adrenergic inhibitory neurotransmission by a negative feedback mechanism were without success.


Correlation between various cell-surface antigens induced by murine leukemia virus in the rat: serological analysis.

Analysis based on the cytotoxicity test using antisera produced in rats indicated the presence of various cell-surface antigens on the tumors in WKA/Mk rats induced or artificially infected by Friend, Rauscher or Gross viruses. Each line of the tumor was found to have a common antigen, namely group-specific cell-surface antigen. A Gross type-specific and Friend and Rauscher type-specific cell-surface antigens were also detected, in keeping with the observations in the mouse system. In addition, the present experiments strongly suggested that a Friend strain-specific antigen was detected only on the cell-surface of the tumors in WAK/Mk rat induced or artificially infected by Friend virus. These results were in parallel with the data of previously reported transplantation studies.


Isolation and partial characterization of an arginine-rich apolipoprotein from human plasma very-low-density lipoproteins: apolipoprotein E.

A water-insoluble apoprotein was isolated from apo-VLDL by column chromatography on Sephadex G-200 in sodium dodecylsulfate followed by preparative polyacrylamide gel electrophoresis in a discontinous sodium dodecylsulfate system, or by preparative electrophoresis alone. The protein was similar in amino acid composition to the "arginine-rich protein" reported by Shore and Shore. It represented about 10% of the total protein mass of VLDL. The apoprotein showed one single band with an apparent Mr of 39000 in sodium dodecylsulfate gel electrophoresis, and was homogeneous in gel electrophoresis at pH 8.9 In 8M urea. Immunochemical studies also showed homogeneity of this protein, and antisera prepared against it did not react with any other of the well known apolipoproteins, but did react with VLDL and apo-VLDL preparations. Analytical isoelectric focusing in 8M urea resulted in a heterogeneous banding pattern showing three major polypeptides with pI values of 5.5, 5.6 and 5.75. Thus this apolipoprotein clearly differs from the apo-B and apo-C polypeptides of VLDL as well as from apoproteins A and D in its molecular weight, amino acid composition, focusing behavior and immunochemical properties.


Studies on the asymmetric arrangement of membrane-lipid-enveloped virions as a model system.

Lipids of BHK 21 cells (baby hamster kidney) grown in tissue culture were labelled with radioactive fatty acids. The enveloped vesicular stomatitis virus was propagated in this host cell type. The virions were purified by density gradient centrifugation. Neuraminidase treatment of the intact virions led to a complete transformation of hematoside [N-acetylneuraminosyl(alpha2-3)lactosyl(beta1-1)ceramide] into lactosylceramide, with identical labelling of the ceramide portion in hematoside of the untreated virions and the lactosylceramide of the neuraminidase-treated particles. The morphology of the virions appeared unchanged in electron micrographs, but the neuraminic-acid-free virions had a strong tendency to aggregate. The results of these studies are evidence that gangliosides are integrated exclusively into the outer lamella of the lipid bilayer in the viral envelope. It is also evident that the viral envelope is a suitable model for studies on membrane asymmetry.


Identification of cytochrome a and a3 in yeast cells.

1) Cells of Saccharomyces cerevisiae have been analysed by single and double-bean spectroscopy. Evidence is given for two components of cytochrome c oxidase in the alpha-region of their absorption spectrum. A rapidly reduceable component with a maximum at 600 nm and a slowly reduceable component with a maximum at 604 nm contribute about equal amounts to the total alpha-absorption of cytochrome c oxidase. 2) The component absorbing at 600 nm was identified as the high-potential component with a redox potential of 340 - 355mV, and the 604-nm component as the low-potential component of cytochrome c oxidase with redox potential of 180 - 190 mV. 3) Both components can be characterized by analysing the reduction kinetics in the presence of carbon monoxide. In the presence of saturating concentrations of carbon monoxide, an oxygen pulse leads to a rapid oxidation and subsequent reduction of cytochrome c oxidase, but the rapid reduction phase at 600 nm completely disappears, demonstrating its identity with cytochrome a3, which, being liganded by carbon monoxide in its reduced state, cannot react any more. The component which becomes oxidized and later reduced in the presence of carbon monoxide -- by definition cytochrome a -- has an absorption maximum at 604 nm. 4) The total extinction change at 604 nm in the presence of carbon monoxide is nearly as high as in its absence, but the reduction occurs in two phases and only the second phase, which contributes 50 - 60% to the total absorbance, corresponds in redox potential and kinetic properties to cytochrome a. Because the redox potential of the first reduction phase is very close to that of the low-potential copper atom of cytochrome c oxidase, it is concluded that the apparent increase in the extinction coefficient of cytochrome a in the presence of carbon monoxide is the result of a strong interaction between the ligand fields of cytochrome a and copper, induced by the binding of carbon monoxide to reduced cytochrome a3.


Short-term follow-up after brief inpatient treatment.

A total of 110 patients admitted to the inpatient unit of a community mental health center in Philadelphia were followed up between 90 and 120 days after discharge to determine their level of functioning after an average hospital stay of 21 days. All but five of the patients were referred for aftercare, and 80 received outpatient or day hospital care after their release. Sixteen patients were readmitted to inpatient care within 90 days after discharge. The author describes the patients' social and community adjustment at follow-up, based on information obtained from aftercare therapists.


Short-term versus long-term hospitalization in a private psychiatric facility: a follow-up study.

A follow-up study was made of 43 short-term and 42 long-term ex-patients 18 months after their discharge from a private psychiatric hospital. The short-term sample was different from the long-term sample on many preadmission and hospitalization variables, but it was much less different on the follow-up variables. Although most ex-patients in both samples were functioning well at follow-up, the long-term sample was doing somewhat better in all areas, according to both the former patients and their nearest relatives. Differences between the improvement of those in the short- and long-term sample increased when tested by diagnosis; long-term schizophrenic patients improved the most.


A follow-up study of clients discharged from a day hospital aftercare program.

Sixty-one former clients of a day hospital aftercare program were assessed for level of social and individual functioning. They were found to be more like psychiatric patients than like the normal population. Marital status and type of residential arrangement were the only demographic variables that could be correlated with level of functioning. Married clients functioned better than those who were not married, and boarding-home residents were significantly more impaired than those in other living situations. Although the data did not permit conclusions about the effectiveness of the aftercare program, they suggested the need for improved conditions in boarding homes.


Giant cell carcinoma of the lung. A light and electron microscopic study.

Three cases of giant cell carcinoma of lung and six other lung carcinomas with large atypical cell formation were studied by both light and electron microscopy. The giant cell carcinomas showed unique light and electron microscopic features, whereas large atypical cells from other lung carcinomas usually retained some ultrastructural characteristics of their cellular origin. The giant cell carcinoma is characterized by abundant mitochondria, a concentric whorl of tonofilament-like fibrils, and aggregates of several pairs of centrioles. The "phagocytic activity" of the giant cell carcinoma is more likely attributable to the so-called tumor cell-tumor cell or leukocyte-tumor cell emperipolesis. Giant cell carcinoma of lung may originate from a primitive multipotential cell in the distal bronchiole or be associated with a squamous, glandular, or clear cell pattern. Whether in pure or mixed form, this tumor should still be considered a specific entity because of its unique light and electron microscopic features and the fulminant clinical course it follows despite rate reported exceptions.


Insect cell culture: improved media and methods for initiating attached cell lines from the Lepidoptera.

Several cell lines from the pupae of the noctuid moth species Spodoptera frugiperda, Heliothis zea, and Trichoplusia ni were isolated on a synthetic medium containing insect hemolymph and turkey serum. These lines were progressively adapted to improved media free of insect hemolymph but containing one or more of the following sera: turkey, chicken, and fetal calf. Primary culture tissue disruption was improved by substituting collagenase for trypsin. Primary culture survival was improved by controlling the total tissue volume per unit medium volum, and by the addition of glutathione to prevent melanization and to improve cell adherence to the substrate. Culture servival was also improved by heat treatment of sera, control of medium osmolality, and changes in the basal medium and serum supplementation. Some of these changes also resulted in improved growth giving higher maximal cell counts. Comparative cell growth on the various media was graphed and generation times given.


Influence of culture conditions of growth of FL-74 cells and feline oncornavirus cell membrane associated antigen production.

The FL-74 cell, a feline lymphoblastoid cell line derived from a tumor induced by leukemia virus, grows equally well in static suspension culture (plastic T-flask or silicone treated glass bottles) or in spinner culture. No growth was observed in unsiliconized glass bottles. Although feline leukemia virus production was nearly the same in FL-74 grown in each of the above types of vessel, the expression of the feline oncornavirus membrane associated antigen (FOCMA), as determined by membrane immunofluorescence, was more intense and more complete on cells grown in static suspension. Moreover, higher fluorescent antibody titer endpoints were observed with cells from static suspension cultures than with cells from spinner cultures, FL-74 cells grown in spinner culture, when subjected to partial synchrony by cold block or by deprivation of essential amino acids (arginine and/or isoleucine) for 12 hr, achieved a membrane fluorescent pattern for FOCMA similar to cells grown in static suspension. It is proposed that the expression of FOCMA on the cell membrane surface is cell-cycle dependent, and that the rate at which a cell passes through the cell cycle determines the pattern and intensity of the fluorescence of the cell membrane.


The positive control of cell proliferation by the interplay on calcium ions and cyclic nucleotides. A review.

Calcium, cyclic AMP, and cyclic GMP do not seem to be involved in proliferative activation of postmitotic differentiated cells. Instead, they are intracycle regulators, and we propose the following working model of their control of the initiation of DNA synthesis. While a role for cyclic GMP cannot yet be defined, a brief postmitotic burst of its synthesis might serve to prevent certain activated cells (e.g. 3T3 mouse cells) from being diverted into a nonproliferating (but still activated) G0 state (Figs. 1 and 17). In a latter part of the G1 phase, something happens to stimulate briefly the synthesis of cyclic AMP which, in turn, drives calcium ions from the mitochondria into the  cytosol to activate newly synthesized thymidylate synthetase (or other primed enzymic assemblies) (Fig. 1). Having "turned on" their target enzymes, the accumulated cyclic AMP is destroyed and the excess calcium ions are reaccumulated by the mitochondria to avoid interfering with succeeding reactions. This model predicts that persistent changes in cyclic AMP metabolism and the respiration-linked, calcium-accumulating (ion-buffering) activity of mitochondria may be responsible for the sustained growth of tumors.


Organ culture of human nervous system tumors.

Tumors of the human nervous system (neuroblastomas, an ependymoma, a medulloblastoma, and a Schwannoma) obtained during surgery have been cultured organotypically by the method of Wolff. The tumors retained characteristic morphology, organization and patterns of behavior in vitro, and one neuroblastoma gave rise to a growing long-term culture. Long-term organotypic culture, where maintenance of tissue organization and growth occur together, is recommended for the study of neoplasms of the nervous system.


Fine structure of a murine mammary carcinoma cell line.

A fine structural study was made of cells from the epithelioid MCF-8/5-2A cell line derived from a MuMTV-free D2 transplantable hyperplastic outgrowth. Electron microscopy shows the cells to be truly epithelial with many cell-to-cell junctions and microvilli. The cells are similar in many respects to normal mouse mammary gland and some of the conventional mammary tumor derived cell lines. This study supports previous observations of the absence of MuMTV in MCF-8 within the limits of morphological detection, and demonstrates the presence of numerous virus particles within, or budding into, cisternae of the endoplasmic reticulum and nuclear envelope. These intracisternal A particles have not been previously described in such abundance in mammary tumor tissue culture cells.


Elevation of adenosine 3',5'-cyclic monophosphate in established mamalian cell strains by Hypaque (sodium diatrizoate).

Hypaque (sodium diatrizoate), when added to cells growing in culture (two HeLa S3 strains, rat glioblastoma C6 and mouse lymphoma L5178Y), increased the intracellular level of adenosine 3',5'-cyclic monophosphate. A transient elevation of adenosine 3',5'-cyclic monophosphate was also observed when L5178Y cells were subjected to a procedure recommended for separation of lymphocytes from peripheral blood. The effect of Hypaque did not appear to be related to the increase in osmolality of the medium.


Fine structural and cytochemical studies of human diploid fibroblasts arrested in an essentially nonmitotic state.

Human diploid fibroblasts can be maintained in vitro in an arrested, essentially nonmitotic state for extended periods of time by reducing the serum concentration in the medium from 10 to 0.5%. Arrested cells can be induced to re-enter the proliferative state by subcultivation in medium containing 10% serum. Fine structure, acid phosphatase, cytochrome oxidase, and extracellular carbohydrates in arrested cells were examined and compared to cultures growing in 10% serum and to cells transferred to 10% serum after 21 days in 0.5% serum. Cells in 10% serum posessed a well-developed Golgi complex, extensive rough endoplasmic reticulum, mitochondria containing transverse cristae, and many free ribosomes in the cytoplasm. In arrested cells, Golgi complexes were rarely observed, the number of both free and membrane-bound ribosomes was reduced, the number of cristae per mitochondria was decreased and the amount of demonstrable cytochrome oxidase activity was diminished. There was an accumulation of intercellular carbohydrate components. After subcultivation with medium containing 10% serum, arrested cells regained the ultrastructural characteristics of cells continuously cultured at this serum level; however, the amount of intercellular carbohydrate remained elevated. These results indicate that distinct yet reversible changes occur in the subcellular morphology and organization of cells maintained in an essentially nonmitotic state. This arrested state may be a close approximation to the situation as it occurs in vivo in expanding cell populations.


Detection of bovine viruses in fetal bovine serum used in cell culture.

This investigation employed a viral screening method detect endogenous bovine virus contaminants in commercially supplied fetal bovine serum. Fifty-one lots of fetal bovine serum from 14 suppliers were examined. Over 30% of the lots tested were found to contain bovine viruses; they included bovine virus  diarrhea virus, parainfluenza type3-like virus, bovine herpesvirus-1, bovine enterovirus type 4, and an unidentified cytopathogenic agent. Of the 51 lots, 20 had been pretested by the suppliers and were considered to be free of known viral contaminants. Our viral screening methods revealed that five of these pretested lots, or 25%, contained endogenous bovine viruses.


Interaction of cholera enterotoxin with cultured adrenal tumor cells.

In the adrenal tumor cell system ganglioside Gm1 inhibited cholera enterotoxin (CT)-induced steroidogenesis if it was preincubated with the toxin or added to adrenal cells 10 min before CT. In the preincubation studies a molar ratio of Gm1 to toxin of 3:1 was necessary for half-maximal inhibition of steroidogenesis. On the other hand, horse serum anticholeragenoid neutralized the steroidogenic response to cell-bound CT by 50% if it was added to adrenal monolayer cultures 15 min after the toxin. Specific antiserum was able to neutralized 20% of the toxin-induced activity even if it was added to adrenal cultures 2 h after CT. Phase contrast microscopy demonstrated that partial neutralization of the biochemical effect of CT by horse serum anticholeragenoid was accompanied by partial prevention of toxin-induced rounding of adrenal cells. Further studies showed that pretreatment of cultured adrenal cells with a maximal dose of CT increased cyclic adenosine 3'-5'-monophosphate formation in response to a maximal stimulating dose of adrenocorticotropin. This result suggested potentiation of hormonal activation of adenylate cyclase in intact adrenal tumor cells in response to CT.


Fate of human lactoferrin and myeloperoxidase in phagocytizing human neutrophils: effects of immunoglobulin G subclasses and immune complexes coated on latex beads.

Human neutrophils (PMN) degranulated in response to soluble human immune complexes and to myeloma proteins, including subclasses of immunoglobulin G (IgG)1, IgG2, IgG3, and IgG4 coated on 1.09-mum latex beads. Immunochemical measurement of lactoferrin (LF) from specific granules and myeloperoxidas (MPO) from azurophil granules showed that both classes of granule degranulated. Beads with soluble complexes of human anti-pigeon IgG-normal pigeon IgG, prepared from serum of a patient with pigeon breeders disease, induced significantly greater degranulation than did pigeon IgG-coated beads. Up to 40% of LF in the PMN degranulated during phagocytic challenge and 86% of that entered the extracellular fluid. Twenty to 30% of the MPO degranulated, but less than 50% of that entered the extracellular fluid. The degranulated LF and MPO which remained in the PMN were recovered from phagocytic vacuoles. Beads coated with purified human myeloma proteins (12 different ones, three of each subclass) induced degranulation in the order IgG3 greater than IgG1 greater than IgG2 greater than IgG4; however, these differences were found to be a function of the amount of latex ingested. Thus, the amount of degranulation was dependent more on the opsonizing capacity of the immunoglobulins rather than on their intrinsic capacities for inducing degranulation. Degranulation of both LF and MPO in response to IgG subclasses followed patterns similar to those caused by soluble immune complexes, and IgG3 coated on beads caused degranulation equal to that caused by human complex-coated beads. Degranulation to IgG3 and IgG4 was uninfluenced by fresh compared with heat-inactivated human AB serum. This was true although IgG3 beads fixed greater than sixfold more complement than did IgG4 beads. Evidently human IgG subclasses enhance phagocytosis and degranulation of human PMN. The overwhelmingly extracellular degranulation of LF in response to various bead coating suggest that it subserves a major protion of it role outside PMN.


Effect of staphylococcal protein A on complement-potentiated neutralization of herpes simplex virus and immune lysis of virus-infected cells.

Interaction of staphylococcal protein A (SPA) with human serum depressed the ability of such serum to neutralize herpes simpled virus (HSV)-antibody labialis. SPA-induced depression of serum-dependent virus neutralization appeared to be due to consumption of complement by SPA. In addition, SPA attached to antibody-treated, HSV-infected cells and inhibited complement mediated immune cytolysis. The amount of inhibition obtained depended upon the with the infected cells. The possible significance of SPA in the pathogenesis of viral disease complicated by secondary staphylococcal infection is discussed.


Murine cytomegalovirus: induction of and sensitivity to interferon in vitro.

Moderate amounts of viral inhibitor were produced by mouse embryo (ME) cultures infected with two strains of plaque-purified murine cytomegalovirus (MCV). This inhibitor was shown to be interferon, based on the possession of similar properties. The growth studies of MCV in ME cells showed that interferon was produced as early as 4 h after infection, infectious virus was produced between 12 to 16 h, and cytopathic effect was produced between 16 to 18 h. Since MCV-induced interferon production and the subsequent development of antiviral state occurred early, the long eclipse period may be due to an interferon-mediated delay of virus replication. Pretreatment of ME cells with varying concentrations of interferon before infection with MCV did not result in increased interferon production, but at high pretreatment doses a slight inhibitory effect on interferon production was observed. In vitro sensitivity studies showed that small doses of MCV were highly sensitive to the antiviral action of interferon, but higher viral doses proved to be markedly resistant. Although the available evidence does not permit a definitive interpretation of the mechanism by which MCV may show differing sensitivities to interferon action, the presence of a small interferon-resistant fraction of virus-infected cells may account for the observations.


Inhibition of the mitogenic response of normal peripheral lymphocytes by extracts or supernatant fluids of a Herpesvirus saimiri lymphoid tumor cell line.

A Herpesvirus saimiri-infected marmoset lymphoid cell line (MLC-1) was examined for the presence of soluble factors which might affect lymphocyte functions and, therefore, relate to the pathogenesis of lymphoma in vivo. MLC-1 cells, cell extracts, and culture fluids were shown to reduce the spontaneous deoxyribonucleic acid (DNA) synthesis of normal peripheral blood lymphocytes and to completely inhibit their response to phytohemagglutinin (PHA). Suppression of PHA response was demonstrated against a variety of human and nonhuman primate species, with 90 to 95% inhibition occurring at dilutions of extract as great as 1:5,120. Inhibition of this type was also demonstrated using extracts of two of five other lymphoblastoid cell lines tested. Physical-chemical characteristics of the active factor(s) revealed a non-sedimentable, non-dialyzable, trypsin-resistant molecule, which was stable at 56 C for 30 min but inactivated at 80 C for 30 min. The factor(s) also exerted an effect on some but not all established lymphoblastoid cell lines, where DNA, ribonucleic acid, and protein synthesis were all inhibited, with DNA synthesis being the most affected (95% suppression). Cellular respiration was not affected by the presence of the factor(s), and the inhibition of DNA synthesis was reversible after 24 h. Purified human interferon did not reduce the PHA response of normal owl monkey peripheral blood lymphocytes and was less effective against an established lymphoblastoid cell line than the MLC-1 extract. Antiviral activity was also demonstrated in the preparations and may represent interferon, which these cells are known to produce at low levels.


Effect of pretreatment with Mycobacterium bovis (strain BCG) and immune syphilitic serum on rabbit resistance to Treponema pallidum.

Stimulation of the rabbit reticuloendothelial system with viable Mycobacterium bovis (strain BCG), and other agents, had no effect on the development of syphilitic lesions after intradermal or intravenous inoculation with graded doses of Treponema pallidum (virulent Nichol's strain; mean infective doses less than 10). The simultaneous administration of immune syphilitic rabbit serum retarded the development of lesions, but this appeared to be due solely to the immune serum, suggesting no synergism between the activated reticuloendothelial system and the anti-T. pallidum antibodies. The administration of two doses of BCG enhanced syphilitic lesion development in the rabbit.


Effect of cyclophosphamide on the response of chickens to a virulent strain of Marek's disease virus.

The effect of cyclophosphamide on the pathogenesis of Marek's disease was examined in a line of chickens which is relatively resistant to Marek's disease. The injection of cyclophosphamide into newly hatched chickens delayed and reduced viremia and also reduced the development of Marek's disease lesions until 2 weeks after exposure to Marek's disease virus. The data indicate that a population of T cells susceptible to infection with virus and possibly viral transformation is affected by cyclophosphamide.


Viral replication and interferon production in fetal and adult ovine leukocytes and spleen cells.

Peripheral blood leukocyte and spleen cell cultures derived from adult sheep and from third-trimester (107 to 145 days of gestation) and second-trimester (70 to 98 days of gestation) fetal lambs were examined for their ability to support viral replication and to produce interferon. Bluetongue virus, Herpesvirus hominis type 2, and Chikungunya virus failed to replicate in either leukocyte or spleen cell cultures derived from adult ewes or in cultures from second- or third-trimester fetal lambs. Similarly, peripheral blood leukocytes from adult sheep or third-trimester fetal lambs did not support the replication of Semliki Forest virus, vesicular stomatitis virus, Newcastle disease virus, or vaccinia virus. No major differences were observed in the ability of fetal and adult leukocytes to produce interferon in response to viral infection. In contrast, mean interferon titers induced by bluetongue virus, H. hominis type 2, and Chikungunya virus in spleen cells from second-trimester fetuses were 4- to 10-fold greater than those induced in spleen cells from adult ewes. Variations in interferon levels induced on separate occasions with cells from the same donor age group were observed. The antiviral substance induced in both the fetal and adult cell cultures fulfilled the usual criteria for characterization as interferon.


Neuropathological effects of persistent infection of mice by mouse hepatitis virus.

Mouse hepatitis virus (MHV3) can persist for months in strains of mice with genetically controlled "semisusceptibility" to this virus. The pathology of the chronic neurological disease induced in these animals has been investigated by conventional histology and immunofluorescence. A2G mice develop a chronic choroidoependymitis and meningitis leading to severe hydrocephalus and hydromyelia. In C3H mice a widespread vasculitis was observed, with both viral antigens and bound immunoglobulins in vessal walls. No significant glomerulonephritis was found. Systemic amyloidosis was present in the spleen, liver, and kidneys. The virus was not detected in neural tissues, but brain and spinal cord lesions were found near inflammatory areas surrounding damaged vessels. It is suggested that viral persistance in ependymal cells is directly responsible for the lesions in A2G mice, whereas an immunopathological lesion of blood vessels of the central nervous system underlines the damage to mice of the C3H strain.


Histopathological effect of Clostridium perfringens enterotoxin in the rabbit ileum.

Highly purified enterotoxin from Clostridium perfringens was found to have histopathological activity in the rabbit ileum. Unlike the action of cholera, Escherichia coli, and Shigella enterotoxins, epithelium was denuded from the tips of ileal villi at concentrations of the enterotoxin necessary to induce fluid accumulation in the rabbit. Whether or not this observed histopathology is essential for the diarrheal syndrome associated with Clostridium perfringens food poisoning remains unclear.


Monocyte activation in horses persistently infected with equine infectious anemia virus.

The monocytes of horses infected with equine infectious anemia virus were shown by their failure to migrate from capillary tubes and their increased adherence to erythrocytes to be activated.


Virus-induced diabetes mellitus. V. Biological differences between the M variant and other strains of encephalomyocarditis virus.

The M variant of encephalomyocarditis virus is very closely related serologically to five other strains of encephalomyocarditis virus. Despite the serological relationship, these five viruses differ markedly from the M variant in their tissue tropisms and only the M variant infects beta cells of the pancreas, producing diabetes.


Origin of lymphoid lines established from mixed cultures of cord-blood lymphocytes and explants from infectious mononucleosis, Burkitt lymphoma and healthy donors.

Lymphocytes were explanted from EBV-seropositive donors including peripheral blood of infectious mononucleosis patients, healthy donors and EBV-genome-carrying cells from Burkitt lymphoma (BL) biopsies or nude mouse-passaged, BL-biopsy-derived lines. The explanted cells were mixed with fresh cord-blood lymphocytes from mice of the opposite sex. In all categories of derived lines, cord-blood cell progeny was predominant, as judged by the sex marker and other associated markers. Only one BL biopsy line, serially passaged in nude mice, gave rise to a monoclonal lymphoma line.


Sensitive method to detect non-complement-fixing antibodies to Epstein-Barr virus nuclear antigne.

The anti-complement immunofluorescence (ACIF) test was modified to detect non-complement-fixing antibodies to Epstein-Barr (EB) virus nuclear antigen (IBNA). These EBNA antibodies belong to the immunoglobulin classes IgA and IgG. In our method anti-human gamma-globulin (AHG) was bound to the EBNA antibodies before the complement was added. If only non-complement-fixing antibodies are present the complement can be fixed to the AHG. Within only a few weeks of the onset of infectious mononucleosis (IM) the non-complement-fixing EBNA antibodies reach high titers while the complement-fixing antibodies (detected by the ACIF test) are not yet present. Anti-EBNA-IgM antibodies were not found in the IgM fractions of sera taken at different stages of IM.


Some electrical properties of the peripheries of murine 3T3 cells with respect to viral transformation and reversion.

Mouse 3T3, Simian virus 40 transformed 3T3 cells (SV3T3) and two SV3T3 lines showing reversion of their transformed phenotype (Rev 3 and Rev 5) have been studied with respect to electrophoretic mobilities and colloidal iron hydroxide (CIH) binding density visible by electron microscopy, before and after incubation with neuraminidase or ribonuclease. The results show that, in general, the marked changes in both sets of surface parameters associated with transformation are largely reversed in the Rev 5 revertant, and only partially reversed in the Rev 3 line. It was also observed that, in common with Ehrlich ascites tumor (EAT) cells examined previously, the densities of CIH-particles bound over the microvilli of all the cell types was 1.5 to 2.7 times higher than those bound to the spaces between them. In contrast to the EAT cells, the higher density of CIH particles bound over the microvilli was not due to neuraminidase-sensitive binding sites.


Studies of genetic transmission of mammary tumour virus by C3Hf mice.

By radioimmunoassay (RIA) mammary tumour virus (MTV) antigens were detected in individual milk samples of C3Hf mice, (female BALB/c X male C3Hf)F1 mice and (female C3Hf X male BALB/c)F1 mice; milk samples of BALB/c mice were negative. In the segregating backcross I population, female BALB/c X male (female BALB/c X male C3Hf) viral antigens were found in the milk of 93 out of 169 mice (55%). In the Bc II population (daughters of Bc I mothers and BALB/c fathers) two groups were distinguished. In the first group, derived from positive Bc I mothers, 55 out of 110 mice (50%) had detectable levels of viral antigens in the milk. In the second group, progeny of negative Bc I mothers, 1 mouse out of 47 was positive. These data are consistent with the assumption that one dominant gene is responsible for the presence of viral antigens in the milk of C3Hf mice. This gene (Mtv-1) seems to be linked with the albino locus situated on chromosome 7; the recombination percentage was about 29. In the first experiment with Bc I mice a significant difference was found between the tumour ages of the mice with virus-positive milk and of the mice with virus-negative milk: all mice (18) with viral antigens in the milk developed mammary tumours at an age ranging from 7 to 18 months, whereas in only 7 out of 16 mice with virus-negative milk were mammary tumours found before the age of 21 months. Viral antigens were detectable (by RIA) in the tumours of mice of both subgroups; however, the amounts (mU/mg tumour) were significantly lower in the tumours derived from mice with virus-negative milk. Although MTV-L of C3Hf mothers could be transmitted to BALB/c mice by foster-nursing, viral antigens could not be detected in milk samples collected prior to the third lactation period; thus an influence on the data of extrachromosomally transmitted MTV-L is unlikely.


Detection of virus-specific DNA and RNA base-sequences in individual cells transformed or infected by adenovirus type 2.

By means of 3H thymidine-labelled adenovirus 2 DNA, adenovirus-specific DNA sequences have been localized in individual nuclei of adenovirus 2- or 12-infected cells, and adenovirus-specific RNA sequences have been detected in the permissive cells as well as in cells transformed by adenovirus type 2. The ability to detect such specific virus RNA base-sequences in particular, by in situ hybridization, should be useful in studying the transcriptional specificities of tumours and neoplasms.


Radical formation in salts of pyrimidines. I. 1-methyluracil. HBr crystals.

Radicals produced by X-irradiation at 77 K of 1-methyluracil. HBr crystals have been analysed by electron spin resonance spectroscopy. The results are compared with those obtained on 1-methyluracil crystals. Four radicals have been identified, two of which are present only in 1-methyluracil. HBr crystals: the C6-addition radical and the pyrimidine--anion radical. Warming-up experiments have been performed in order to study the secondary radical reactions. The different mechanisms proposed for the radiolysis of DNA constituents in the solid state are discussed in connection with these findings.


E.s.r. studies of sulphur-substituted pyrimidines. 2-thio-5-carboxyuracil at 77 K.

Single crystals of 2-thio-5-carboxyuracil were irradiated and studied at 77 K with e.s.r. spectroscopy. Five resonances were observed and related to the sulphur atom in the 2 position of the pyrimidine ring. Three of the resonances have been assigned to three conformations of a radical formed by hydrogen abstraction from N1. The principal values for the nitrogen coupling are 9-7, 0-0 and 0-0 gauss. The g tensor principal values are 2-173, 1-997 and 1-990 for the dominant conformation of this radical. Two other radicals could not be identified unambiguously.


Ionic base radicals in gamma-irradiated oriented non-deuterated and fully deuterated DNA.

The free radicals induced by gamma-irradiation at 77 K in non-deuterated and fully deuterated oriented samples of moist algal DNA have been studied by electron paramagnetic resonance (e.p.r.). The e.p.r. spectra from the non-deuterated sample were found to be similar to those from calf-thymus DNA studied previously. Numerical spectra simulations were performed based on the previously proposed interpretation of the DNA spectra at 77 K in terms of a mixture of two spectral components arising from anionic and cationic base radicals (probably on thymine and guanine, respectively). The simulations were found to account satisfactorily for the main characteristics of the e.p.r. spectra from both the non-deuterated and the fully deuterated algal DNA sample.


Consultation with the filipino boarding home: an after-care facility in Hawaii.

This report describes a unique Hawaiian after-care facility the Filipino operated boarding home, and the approaches useful in consultation with the operators. The majority of the boarding homes for psychiatric patients are operated by recent Filipino immigrants. This fact is explained by their current social position and also by cultural values--such as aiding others and an extended family system--which are present in the Philippines. In consultation with seventeen such boarding home operators, cultural beliefs and values played a great part in their approach to patients. These approaches created special problems and assets in their management of patients from other ethnic groups such as Japanese, Caucasian and Hawaiian. A problem-oriented approach to consultation proved useful initially and set the stage for other forms of intervention. This included allowing the operator to ventilate her feelings about the patient; bridging the communication gap between operators and patients; dissipating the operators' stereotypic perception of patients; and educating the operators about mental illness. Our impressions and indirect evidence indicated that Filipino boarding home operators perform a useful service and, with consultation sensitive to their values, can become even more effective.


DNA replication in SV40-infected cells. XI. The properties of SV40 DNA and nucleoprotein complex synthesized in the presence of cycloheximide.

Cycloheximide inhibits simian virus 40 (SV40) DNA synthesis in productively infected African green monkey kidney cells. The residual viral DNA synthesized in the presence of this drug is deficient in superhelical turns as measured by band sedimentation in the presence of several concentrations of ethidium bromide. The superhelical density of SV40 DNA synthesized in the presence of cycloheximide is about one half that of viral DNA produced in the absence of this drug. Ribonuclease H and alkali treatment were employed to determine if ribonucleotides could be detected in closed circular SV40 DNA synthesized in the presence of cycloheximide. While both these approaches detected ribonucleotides in mitochondrial DNA, no identification of ribonucleotides in SV40 DNA synthesized in the presence of cycloheximide was found. The SV40 nucleoprotein complex synthesized in the presence of cycloheximide has a slower sedimentation rate (35S) and a higher bouyant density (1.54 g/cm3) than the complex made in the absence of this drug (50S; 1.47 g/cm3). The protein:DNA ratio of the SV40 nucleoprotein complex is normally about 0.9. This ratio is reduced to about 0.6 when the complex is synthesized in the presence of cycloheximide.


Cyclic appearance of defective interfering particles of herpes simplex virus and the concomitant accumulation of early polypeptide VP175.

Serial passage of undiluted herpes simplex virus types 1 and 2 resulted in cyclic production of infectious and defective virions. Defective virus production was characterized by the appearance of a new species of viral DNA with a higher bouyant density in CsCl than standard viral DNA. Measurement of the infectivity titer and DNA synthesis revealed that the defective particles interfered with the replication of standard virions and stimulated the overproduction of a large molecular weight (175,000 daltons) polypeptide.


Characteristics of infection by RSV. III. Duration of the latent period in unsynchronized chick embryo fibroblasts.

The duration of the latent period of RSV was studied in unsynchronized chicken cells using two methods. First, RAV1-producing cells were infected with Schmidt-Ruppin strain of Rous sarcoma virus (SR-RSV;subgroup D), and new virions of SR-RSV (subgroup A) produced after phenotypic mixing with RAV1 were neutralized with an antiserum to RAV1. Secondly, uninfected chicken cells were infected with SR-RSV (D) in the presence of either arabionsylcytosine, actinomycin D or cycloheximide, to inhibit the virus production. In both methods, the early decrease of the virus titer showed that new virions were produced as early as 45 min after the beginning of the infection. Earlier times were not explored.


An analysis of factors which influence the local accumulation of bone-seeking radiopharmaceuticals.

A thoretical consideration of the factors which influence the accumulation of radiopharmaceuticals in bone is presented. The avidity with which 99mTc-Sn-pyrophosphate and 18F fluoride are adsorbed by bone crystals has been measured in vitro and the efficiency with which these radiopharmaceuticals are extracted by normal bone has been measured in vivo. It is postulated that alterations in the capillary permeability in the region of a bone lesion greatly influence the target to background ratio that can be obtained with bone seeking radiopharmaceuticals. Quantitiated gamma camera studies in man are presented in support of this hypothesis.


Polypeptide and RNA composition of the reticuloendotheliosis viruses.

The RNA and polypeptide composition of chick syncytial virus (CSV) and duck infectious anemia virus (DIAV) was investigated and compared to that of reticuloendotheliosis virus (REV) strain T, the prototype of the newly recognized REV group of viruses. CSV and DIAV contain genomic RNA species which cosediment with those of REV in sucrose gradients. Five or six polypeptides, two of which are glycoproteins, were consistently found in CSV and DIAV preparations. The major nonglycosylated polypeptides and glycoproteins of CSV and DIAV comigrated with the corresponding polypeptides of REV strain T. Since the genomic RNA species and the glycoproteins of avian tumor viruses fail to comigrate, this suggests that the REV complex is a more homogeneous group.


Spontaneous interferon production and Epstein-barr virus antigen expression in two human lymphoid cell lines and their somatic cell hybrids.

Two human lymphoid cell lines, Raji and Namalwa, and seven somatic cell hybrids between these two cells were tested for spontaneous interferon production. Namalwa and three of the hybrids produce interferon while the four other hybrids, like Raji, do not. Though the Namalwa line is relatively resistant to superinfection with the Epstein-Barr virus (EBV), all hybrids are as susceptible to EBV as the Raji parent. High extracellular concentrations of interferon likewise had little effect on EBV superinfection of Raji or the hybrid cells and it is possible that Raji produces some factor that antagonizes the action of interferon to allow virus expression.


Influence of certain immunodepressants on experimental flavivirus and enterovirus infections in mice.

The effects of cyclophosphamide and 1,3-(piperidinomethyl)-5-phenyl-5-ethylbarbituric acid on certain flavi- and enterovirus infections in mice were studied. Differential enhancement of mortality rates after extraneural and, less markedly, intracerebral virus inoculation was noted. While depression of humoral and/or cell-mediated immunity is considered to be responsible for the effects observed in flavivirus infections, impaired function of the reticuloendothelial system seems to contribute mainly to the potentiation of Mengo virus infection by the immunodepressants used.


Observations on ambulatory electrocardiographic monitoring in clinical practice.

Holter monitoring is a practical technique for determining rate, rhythm, contour, and conduction changes in the electrocardiogram of the ambulatory patient. This kind of information may be of great clinical importance in coronary artery disease with or without myocardial infarction, in patients with permanent pacemakers, in the presence of central nervous system symptoms, and perhaps in patients with chronic obstructive pulmonary disease. Correlating ambulatory electrocardiographic findings in patients with these diseases and syndromes provides insight into the severity of the process involved. It may also define the response to therapy and to specific social and work situations. Present instrumentation, although reasonably effective, may be considered in an early developmental stage. Future progress in terms of miniaturization, automatic patient warning systems, and activation of central recording units, may further broaden the implications for ambulatory electrocardiographic monitoring. The scope of these observations and the variety of unsuspected rhythm and contour abnormalities defined may ultimately rival the information gained in monitored hospitalized patients. It is hoped that this information will have an impact on the therapy of the ambulatory patient comparable to that which coronary-care unit monitoring has had on the hospitalized patient with acute myocardial infarction.


Tracer and freeze-etching analysis of intra-cellular membrane-junctions in Paramecium with a note on a new heme-nonapeptide tracer.

In paramecia the membranes of alveoli and trichocysts are permanently connected to the cell membrane by membrane-junctions, which consist of membrane-intercalated particles in a regular geometrical arrangement. Trichocysts contain secretory material discharged by exocytosis. In unfixed or fixed cells these two compartments were impermeable to the following tracers: To "microperoxidases", i.e. a cytochrome c-derived heme-nonapeptide and a heme-undecapeptide (WM approximately 1650, 1900) applied in vivo, as well as to lanthanum and cytochrome c used during (La) or after (cytochrome c) fixation. The heme-nonapeptide was prepared by TPCK trypsin digestion of cytochrome c and subsequent purification by Sephadex gel chromatography--a simple and inexpensive new procedure resulting in preparations of high yield and purity. Tracers entered alveoli only when the plasmalemma and the alveolar membranes ruptured upon glutardialdehyde fixation. In no case were transmembraneous channels detectable in regions containing membrane-intercalated particles; this holds true for all tracers used and for freeze-fracture replicas obtained by tantalum-tungsten evaporation. With regard to attachment sites over trichocysts our results do not support the assumptions by others according to which exocytosis would be driven by an osmotic shift via transmembraneous channels (which would be analogous to inter-cellular coupling phenomena mediated by gap-junctions), unless such channels would be assumed to operate as carriers rather than via diffusion. Tracers did not penetrate trichocysts before exocytosis occurred. The functional role of membrane-intercalated particles on trichocyst attachments remains unclear. Despite some resemblance with gap-junctions all types of intra-cellular membrane-junctions investigated are functionally "tight" at the level of "resolution" obtained with tantalumtungsten-shadowing and with the tracers used.


Septamycin, a polyether antibiotic. Taxonomy, fermentation, isolation and characterization.

Septamycin is a metal complexing polyether antibiotic produced by a strain of Streptomyces hygroscopicus NRRL 5678. The metabolite, a monocarboxylic acid, was isolated as the sodium salt C48H81NaO16. The crystal structure and absolute configuration were established by X-ray analysis of the p-bromophenacyl derivative. Septamycin has a thirty-carbon backbone and contains seven heterocyclic rings. Supported by direct comparison septamycin proved to be identical with antibiotic A28695 A isolated from Streptomyces albus NRRL 3883. The metabolite is active against gram-positive bacteria and Eimeria tenella (chicken coccidiosis).


Brevibacterium liquefaciens adenylate cyclase and its in vivo stimulation by pyruvate.

Adenylate cyclase of Brevibacterium liquefaciens depends on pyruvate for activity. Growing in a simple medium containing glucose and DL-alanine, the microorganism excreted pyruvate, which reached 20 mM in the medium at stationary phase. Using [3H]adenosine to label the adenosine 5'-triphosphate pool, we showed that pyruvate in the medium stimulated adenylate cyclase of B. liquefaciens in vivo, in a manner similar to the stimulation observed in vitro. Adenylate cyclase in cells harvested at different phases of growth was equally responsive to exogenous pyruvate, indicating that the allosteric site for pyruvate was present in the enzyme throughout the various phases of cell growth. The specific activity of adenylate cyclase was highest in cells harvested at early log phase; thereafter it declined and was substantially lower at stationary phase. Although adenylate cyclase appears to be activated by pyruvate throughout the life span of the cell, the activity appears not to be critical to cell growth, which was comparable whether the medium contained high or low pyruvate.


Operator-constitutive mutants in the threonine operon of Escherichia coli K-12.

Three Escherichia coli K-12 mutant strains resistant to DL-alpha-amino-beta-hydroxyvaleric acid were isolated in which the expression of the thr operon is constitutive. The localization and dominance properties of the mutations involved, called thrO, are those of operator mutations. The gene sequence is OABC as suggested by earlier studies.


Electron-transferring flavoprotein of Peptostreptococcus elsdenii that functions in the reduction of acrylyl-coenzyme A.

In Peptostreptococcus elsdenii, a three-component flavoprotein electron transfer system catalyzes the oxidation of lactate and the reduction of crotonyl-coenzyme A (CoA). Spectral evidence showed that D-lactate dehydrogenase, when reduced by D-lactate, was able to reduce butyryl-CoA dehydrogenase, but only in the presence of the electron-transferring flavoprotein. Reduced nicotinamide adenine dinucleotide could replace reduced D-lactate dehydrogenase. A reconstituted system, containing the three partially purified enzymes, excess D-lactate, and a limiting amount of crotonyl-CoA, reduced the crotonyl-CoA to butyryl-CoA, but only if all components were present. The electron-transferring flavoprotein activity, purified 22-fold, was separated into two major flavoprotein components, A and B, after polyacrylamide gel electrophoresis. Elution of the proteins and subsequent kinetic assays of the eluates showed that component B catalyzes the reduction of butyryl-CoA dehydrogenase by reduced D-lactate dehydrogenase, whereas component A does not. Both A and B catalyzed the reduction of butyryl-CoA dehydrogenase by reduced nicotinamide adenine dinucleotide. The results suggest that the D-lactate dehydrogenase-dependent reduction involves a heretofore unrecognized component of the electron-transferring protein group which may utilize an unusual flavin, 6-hydroxy-7,8-dimethyl-10-(ribityl-5'-adenosine diphosphate)-isoalloxazine.


Multiple regulatory events in the development of competence for genetic transformation in Haemophilus influenzae.

Cyclic adenosine 3',5'-monophosphate derepresses enzyme synthesis but does not yield high levels of transformability or detectable levels of competence-related envelope polypeptides. Additional regulatory events must trigger complete expression of competence.


Competitive inhibition of transformation in group H Streptococcus strain Challis by heterologous deoxyribonucleic acid.

Glucosylated deoxyribonucleic acid (DNA) from phages T4 and T6 competes poorly with homologous DNA causing only a slight decrease of transformation in Group H Streptococcus strain Challis. Other types of heterologous DNAs (Micrococcus luteus, Clostridium perfringens, Escherichia coli, calf thymus and non-glucosylated phage T6 DNA), in contrast to glucosylated T4 and T6 DNAs, compete with transforming DNA to the normal, high extent. These results indicate that as in transformation of Bacillus subtilis, the presence of glucose attached to 5-hydroxymethylcytosine in phage T6 DNA considerably decreases the interaction of such DNA with competent cells of the Challis strain. It also indicates that the guanine plus cytosine content of DNA is not decisive in determining its interaction with competent cells.


Purification and some properties of cytochrome c-552 from a sulfur-oxidizing bacterium, Thiobacillus thiooxidans.

A soluble cytochrome c-552 from Thiobacillus thiooxidans was highly purified and its physico-chemical properteis were studied. The absorption maxima were at 552,523,418 nm in the reduced from and at 412 nm in the oxidized form. The pyridine hemochrome spectrum was the same as that of other cytochromes c. The molecular weight, estimated by the gel filtration method, was found to be 12,600. The isoelectric point was determined to be 9.2-9.3 by the electrofocusing technique. The standard oxidation-reduction potential of this cytochrome was +0.247 V.


Purification of homogeneous glutamine-dependent carbamyl phosphate synthetase from ascites hepatoma cells as a complex with aspartate transcarbamylase and dihydroorotase.

Glutamine-dependent carbamyl phosphate synthetase [EC 2.7.2.9] was purified 1,300-fold from rat ascites hepatoma cells (AH 13) as a multienzyme complex with aspartate transcarbamylase[EC 2.1.3.2] and dihydroorotase[EC 3.5.2.3], using dimethyl sulfoxide, glycerol, and dithiothreitol as stabilizers. The purified complex was essentially homogeneous on agarose-acrylamide composite gel electrophoresis and analytical ultracentrifugation. Its molecular weight was estimated to be about 870,000 by sedimentation equilibrium studies. After alkylation with iodoacetamide or reduction with 0.6% dithiothreitol at 100 degrees, the complex gave a single band on polyacrylamide gel electrophoresis in sodium dodecyl sulfate in a position corresponding to a molecular weight of 210,000. These results indicate that the complex consists of four subunits of similar size.


Role of ubiquinone-10 in electron transport system of chromatophores from Rhodospirillum rubrum.

The role of ubiquinone-10 in the activities for the reduction of free cytochrome c2 and bound cytochrome cc' by succinate was studied with chromatophores from a blue-green mutant (G-9) of Rhodospirillum rubrum. 1. By a single extraction with isooctane, approximately 90% of ubiquinone-10 was easily removed from the chromatophores. In the extracted chromatophores, the activity for succinate-cytochrome c2 reduction decreased to 5-10% of the original activity. This depressed activity was mostly restored by adding ubiquinone-10. The remaining quinone was hardly extractable, even by repeated extractions. With repeatedly extracted chromatophores, the activity for succinate-cytochrome c2 reduction was mostly restored to the same extent as with once-extracted chromatophores, whereas the extent of inhibtion of the activity by antimycin A gradually fell. 2. In isooctane-extracted chromatophores, the activity for the reduction of bound cytochrome cc' by succinate under anaerobic conditions decreased to 35 to 95% of the original level. With chromatophores in which the remaining activity was as low as 40% of the original level, the activity was partially restored by adding ubiquinone-10, but this was not the case with chromatophores in which the remaining activity was higher than approximately 50% of the original level.


Characterization of two soluble ferredoxins as distinct from bound iron-sulfur proteins in the photosynthetic bacterium Rhodospirillum rubrum.

In an earlier investigation (Shanmugam, K. T., Buchanan, B. B., and Arnon, D. I. (1972) Biochim. Biophys. Acta 256, 477-486) the extraction of ferredoxin from Rhodospirillum rubrum cells with the aid of a detergent (Triton X-100) and acetone revealed the existence of two types of ferredoxin (I and II) and led to the conclusion that both are membrane-bound. In the present investigation, ferredoxin and acid-labile sulfur analyses of photosynthetic membranes (chromatophores) and soluble protein extracts of the photosynthetic bacteria R. rubrum and Rhodopseudomonas spheroides showed that ferredoxins I and II are primarily components of the soluble protein fraction. After their removal, washed R. rubrum chromatophores were found to contain a considerable amount of tightly bound iron-sulfur protein(s), as evidenced by acid-labile sulfur and electron paramagnetic resonance analyses. Thus, like all other photosynthetic cells examined to date, R. rubrum cells contain both soluble ferredoxins and iron-sulfur proteins tightly bound to photosynthetic membranes. The molecular weights of ferredoxins I and II from photosynthetically grown R. rubrum cells were found to be 8,800 and 14,500, respectively. Using these molecular weights, the molar extinction coefficients at 390 nm for ferredoxins I and II were determined to be 30.3 and 17.2 mM-1 CM-1, respectively. Ferredoxin I contains 8 non-heme iron and 8 acid-labile sulfur atoms per molecule; ferredoxin II contains 4 non-heme iron and 4 acid-labile sulfur atoms per molecule. Ferredoxin I was found only in photosynthetically grown cells whereas ferredoxin II was present in both light- and dark-grown cells. Ferredoxin II from both light- and dark-grown cells has the same molecular weight (14,500) and absorption spectrum and has 4 iron and 4 acid-labile sulfur atoms per molecule. Low temperature electron paramagnetic resonance spectra of oxidized and photoreduced ferredoxins I and II from R. rubrum were recorded. The EPR spectrum of oxidized ferredoxin II exhibited a single resonance line at g = 2.012. Oxidized ferredoxin I, however, exhibited a spectrum that may arise from the superimposition of two resonance lines near g = 2.012. Photoreduced ferredoxin II displayed a rhombic EPR spectrum with a g value of 1.94. Photoreduced ferredoxin I exhibited a similar EPR spectrum at a temperature of 16 K, but when the temperature was lowered to 4.5 K the spectrum of ferredoxin I changed. This temperature-dependent spectrum may result from a weak spin-spin interaction between two iron-sulfur clusters. These results are consistent with the conclusion that R. rubrum ferredoxins I and II are, respectively, 8 iron/8 sulfur and 4 iron/4sulfur proteins.


Function of sulfhydryl groups in ribosome-elongation factor G reactions. Assignment of guanine nucleotide binding site to elongation factor G.

Titration of elongation factor G (EF-G) with the thiol reagents 5,5'-dithiobis(2-nitrobenzoate) (DNTB), p-hydroxymercuribenzoate (HMB), and N-ethylmaleimide and analysis of cysteic acid after performic acid oxidation revealed a total of four sulfhydryl groups per EF-G molecule. One of these is exposed in the native state and could be used to distinguish between two different conformations of EF-G in our preparations according to its rate of reaction with DTNB and HMB. No evidence for disulfide bridges was obtained. Among the different nucleotides tested, GTP, GDP, and GMP were able to protect the native sulfhydryl group against reaction with DTNB in the absence of ribosomes. Their Kd values with the faster reacting EF-G were 3.4 x 10(-4) M, 0.3 X 10(-4)M, and 2.0 x 10(-4) M, respectively. Because of the specificity of protection by guanine nucleotides and the correspondence of the Kd values with Ki values for GDP and GMP in the ribosome-EF-G GTPase reaction, their binding site on EF-G should be closely related to the active center for ribosome-dependent GTP hydrolysis. Blockage of the native sulfhydryl group of EF-G with a variety of irreversible thiol reagents reduced its activity from one to two-thirds in ribosome-dependent complex formation, GTP hydrolysis, and poly(U)-directed poly(phenylalanine) synthesis. A test of the N-ethylmaleimide-treated EF-G showed both the Km and Vmax of the GTPase reaction to be affected. Thus, the native sulfhydryl group, although important, appears not to be located in the GTPase active center. Denaturation of EF-G with guanidine-HCl and random blockage of any of the three masked sulfhydryl groups caused inactivation, likely due to steric interference with proper chain folding upon renaturation. Treatment of ribosomes or ribosomal subunits with six different thiol reagents at a concentration of 0.27 mM had little or no effect on the ribosome-EF-G GTPase, except for the case with HMB which inactivated the 30 S subunit. An interaction of EF-G with the 30 S subunit in addition to that known to occur with the 50 S subunit is suggested by a rapid and preferential exchange of HMB from the native sulfhydryl group of EF-G to the 30 S subunit of 70 S ribosomes.


Structural requirements for the interaction of 1 alpha, 25-(OH) 2- vitiamin D3 with its chick interestinal receptor system.

The mechanism by which 1 alpha,25-dihydroxycholecalciferol (1alpha,25-(OH)2D3), the biologically active metabolite of cholecalciferol (vitamin D3), stimulates intestinal calcium absorption has been shown to involve an interaction of the steroid with a specific cytosol-chromatin receptor system in this target organ. Thus, 1alpha,12(OH)2D3 binds to a specific cytoplasmic receptor protein and then, following a temperature-dependent step, becomes associated with a finite number of chromatin acceptor sites prior to the initiation of the physiological response. In this respect, 1alpha,25(OH) 2D3 is similar to a number of other steroid hormones. In this investigation, studies were performed to help define the essential structural features required for interaction of 1alpha,25-(OH)2D3 with its intestinal receptor system, and presumably, for biological activity. To this end, competition studies utilizing a series of closely related structural analogs of cholecalciferol were carried out by means of a competitive binding assay highly specific for 1alpha,25(OH)2D3. The competitive binding assay employed in this study is dependent upon the ability to duplicate, in vitro, the conditions which permit the saturable binding of 1alpha,25-(OH)2[3H]D3 to chick intestinal chromatin, in vivo. Optimal conditions for this assay were achieved by the incubation of a reconstituted intestinal receptor system consisting of separately isolated cytosol and Triton X-100 chromatin fractions at 25 degrees for 45 min with 2.0 X 10-8 M 1alpha,25-(OH)2[3H]D3. Maximal binding of about 21 to 24 pmol of radioactive steroid bound per chick intestinal chromatin occurred under these conditions. The ability of the various analogs to compete with the radioactive hormone was assessed by virtue of a decrease in the amount of radioactive steroid bound to the chromatin in the presence of increasing concentrations of nonradioactive analog.


Binding and degradation of 125I-insulin by rat hepatocytes.

The binding and the velocity of degradation of 125I-insulin in the absence or presence of varying concentrations of native procline insulin were studied using isolated rat hepatocytes. At insulin concentrations ranging from 5 X 10(-11) to 10(-6) M, insulin degradation velocity showed a first order dependence on the total concentration of insulin bound at steady state. The overall reaction had an apparent rate constant of 0.030 +/- 0.011 min-1. Furthermore, the degradation of a given amount of 125I-insulin bound to cells was more rapid and extensive than the degradation of the same amount of insulin which had been newly exposed to fresh cells. Mid pretreatment of isolated hepatocytes with trypsin or chymotrypsin at concentrations of 5 to 20 mug/ml depressed to the same degree the amount of 125-I-insulin bound at steady state and the 125I-insulin degradation velocity. Peptide or protein hormones unrelated to insulin, including the oxidized A and B chains of insulin, failed to depress the amount of insulin bound or the velocity of insulin degradation when present at concentrations of 10-5  or 10-6 M. Over a wide range of concentrations, various synthetic insulin analogues and naturally occurring insulins depressed to the same degree the amount of 125I-insulin bound at steady state and the 125I-insulin degradation velocity. These observations suggest that insulin bound to hepatocyte plasma membranes is the substrate for insulin degradation by the liver.


Avian myeloblastosis virus DNA polymerase. Kinetic studies on the incorporation of noncomplementary nucleotides.

The high error rate characteristic of DNA polymerases from RNA tumor viruses has permitted measurements on the simultaneous incorporation of complementary and noncomplementary nucleotides during DNA synthesis. For example, avian myeloblastosis virus DNA polymerase incorporates 1 molecule of dCMP for approximately 500 molecules of dTMP polymerized using polyriboadenylic acid as a template. The parallel incorporation of complementary and noncomplementary nucleotides afer gel filtration of avian myeloblastosis virus DNA polymerase indicates that the observed fidelity is catalyzed by the polymerase itself. Nearest neighbor analysis of the product indicates that noncomplementary nucleotides are incorporated as single base substitutions. The incorporation of the noncomplementary dCMP is not reduced by a 20-fold greater amount of the complementary nucleotide, dTTP. Conversely, the concentration of the noncomplementary nucleotides does not effect the rate of incorporation of the complementary nucleotide. A similar lack of competition between complementary dGTP and noncomplementary dATP is exhibited using poly(rC)-oligo(dG) as a template-primer. Furthermore, there was no detectable competition between the different noncomplementary nucleotides. Possible explanations for this lack of competition are considered.


Amino acid sequence of cytochrome c' from the purple photosynthetic bacterium Rhodospirillum rubrum S1.

The amino acid sequence of cytochrome c' from the purple photosynthetic bacterium Rhodospirillum rubrum S1 has been determined and is consistent with homology to cytochrome c' from the nonphotosynthetic bacterium Alcaligenes sp. NCIB 11015. There is 29% identity in the chosen  alignment of these two proteins. R. rubrum cytochrome c' is composed of a single peptide chain of 126 amino acid residues with a single heme covalently bound near the COOH terminus. There is no sequence similarity to mitochondrial cytochrome c, except at the heme binding site.


Effect of serum on phenylalanine hydroxylase levels in cultured hepatoma cells.

Continued high levels of phenylalanine hydroxylase in cultured H4-II-E-C3 rat hepatoma cells require either serum or glucocorticoids in the culture medium. Upon withdrawal of serum, cellular phenylalanine hydroxylase levels decay exponentially with a half-life of 22 hours for about 60 hours, after which time a low, constant enzyme content persists for at least 96 hours. This decline of phenylalanine hydroxylase is fully reversible; normal enzyme levels are restored in a time- and dosage-dependent fashion upon addition of serum  to basal cultures. The serum factor is nondialyzable and moderately heat-stable. The stimulation by serum of the phenylalanine hydroxylas content of basal cultures is blocked by 3-[2-(3,5-dimethyl-2-oxocyclohexyl)-2-hydroxyethyl]glutarimide and requires ongoing cellular protein synthesis. When added to the enzyme-assay mixture in vitro, serum does not alter the phenylalanine hydroxylase activity of extracts from basal cultures. Three lines of evidence suggest that serum contains a nonsteroidal phenylalanine hydroxylase stimulatory components(s): (a) glucocorticoid antagonists inhibit less than one-half of the biological activity of serum; (b) exhaustive extraction of endogenous serum glucocorticoids with charcoal reduces the activity of serum to about one-half of control values; and (c) the stimulatory effects of charcoal reduces the values; and (c) the stimulatory effects of charcoal-extracted serum and hydrocortisone are additive. The phenylalanine hydroxylase stimulatory activities of the charcoal-extracted sera from four mammalian species and from three stages in development in one mammalian species are comparable. A survey of partially purified preparations of a number of known hormones failed to reveal any one capable of elevating the phenylalanine hydroxylas levels of basal cultures in a manner comparable to that of charcoal-extracted serum.


Role of lysosomal acid lipase in the metabolism of plasma low density lipoprotein. Observations in cultured fibroblasts from a patient with cholesteryl ester storage disease.

The hydrolysis of cholesteryl esters contained in plasma low density lipoprotein was reduced in cultured fibroblasts derived from a patient with cholesteryl ester storage disease, an inborn error of metabolism in which lysosomal acid lipase activity is deficient. While these mutant cells showed a normal ability to bind low density lipoprotein at its high affinity cell surface receptor site, to take up the bound lipoprotein through endocytosis, and to hydrolyze the protein component of the lipoprotein in lysosomes, their defective lysosomal hydrolysis of the cholesteryl ester component of the lipoprotein led to the accumulation within the cell of unhydrolyzed cholesteryl esters, the fatty acid distribution of which resembled that of plasma lipoprotein. When the cholesteryl ester storage disease cells were incubated with low density lipoprotein, the reduced rate of liberation of free cholesterol by these mutant cells was associated with a delay in the occurrence of two lipoprotein-mediated regulatory events, suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity, and activation of endogenous cholesteryl ester formation. In contrast to their defective hydrolysis of exogenously derived lipoprotein-bound cholesteryl esters, the choleseryl ester storage disease cells showed a normal rate of hydrolysis of cholesteryl esters that had been synthesized within the cell. These data lend support to the concept that in cultured human fibroblasts cholesteryl esters entering the cell bound to low density lipoprotein are hydrolyzed within the lysosome and that one of the functions of this intracellular organelle is to supply the cell with free cholesterol.


Saccharopine dehydrogenase. Substrate inhibition studies.

In the direction of reductive condensation of alpha-ketoglutarate and lysine, saccharopine dehydrogenase (N6-(glutar-2-yl)-L-lysine:NAD oxidoreductase (lysine-forming) is inhibited by high concentrations of alpha-ketoglutarate and lysine, but not by NADH. NAD+ and saccharopine show no substrate inhibition in the reverse direction. Substrate inhibition by alpha-ketoglutarate and lysine is linear uncompetitive versus NADH. However, when the inhibition is examined with alpha-ketoglutarate or lysine as the variable substrate, the double reciprocal plots show a family of curved lines concave up. The curvature is more pronounced with increasing concentrations of the inhibitory substrate, suggesting an interaction of variable substrate with the enzyme form carrying the inhibitory substrate. These inhibition patterns, the lack of interaction of structural analogs of lysine such as ornithine and norleucine with the E-NAD+ complex (Fujioka M., and Nakatani, Y. (1972) Eur. J. Biochem. 25, 301-307), the identity of values of inhibition constants of alpha-ketoglutarate and lysine obtained with either one as the substrate inhibitor, and the substrate inhibition data in the presence of a reaction product, NAD+, are consistent with the mechanism that substrate inhibition results from the formation of a dead-end E-NAD+-alpha-ketoglutarate complex followed by the addition of lysine to this abortive complex.


Adenosine 3':5'-monophosphate-regulated phosphorylation of erythrocyte membrane proteins. Separation of membrane-associated cyclic adenosine 3':5'-monophosphate-dependent protein kinase from its endogenous substrates.

An adenosine 3':5'-monophosphate (cyclic AMP)-binding protein in the human erythrocyte plasma membrane was isotopically labeled using a photoaffinity analog of cyclic AMP, N6-(ethyl 2-diazomalonyl) cyclic [3H]AMP. The cyclic AMP-binding site is located in a polypeptide chain having a molecular weight of 48,000. Cyclic AMP-binding protein and cyclic AMP-dependent protein kinase were solubilized with 0.5% Triton X-100 in 56 mM sodium borate, pH 8, but 32P-labeled membrane phosphoproteins were retained in the Triton-insoluble fraction, suggesting that the membrane-associated binding protein is not a primary substrate for protein kinase. Triton-solubilized and membrane-associated protein kinase activities were stimulated 15- and 17-fold by cyclic AMP, suggesting that the degree of association between the catalytic anc cyclic AMP-binding components was very similar in both preparations. Fractionation and characterization of membrane phosphoproteins have shown that protein III and a co-migrating minor protein are substrates for protein kinase but membrane sialoglycoproteins are not phosphorylated.


Role of carbohydrate of human chorionic gonadotropin in the mechanism of hormone action.

The role of the carbohydrate part of human chorionic gonadotropin (hCG) was investigated by measuring the ability of hCG derivatives lacking various sugar residues to bind to rat Leydig cells and stimulate them to synthesize testosterone and cyclic adenosine 3':5'-monophosphate (cyclic AMP). Whereas sequential removal of the sialic acid, galactose, N-acetylglucosamine, and mannose residues led to a progressive increase in the effective dose of the hormone required to stimulate steroidogenesis, it resulted in a marked loss in the ability of the hormone to stimulate cyclic AMP accumulation. Low doses of the glycosidase-treated hormone derivatives were additive with hCG when their ability to stimulate testosterone synthesis was analyzed. Nevertheless, the glycosidase-treated derivatives were potent inhibitors of hCG-induced cyclic AMP accumulation, suggesting that removal of the sugars did not influence binding of the hormone to the cell as much as it reduced the ability of the bound hormone to activate adenyl cyclase. This hypothesis was further supported by our finding that the hCG derivatives were highly effective inhibitors of 125I-hGC binding to the intact cells. Removal of sialic acid and galactose enhanced the inhibition, whereas removal of all the sugar residues only decreased the inhibition slightly. The degree of these effects was comparatively small. The possibility that steroidogenesis and cyclic AMP accumulation are altered independently by hCG stimulation is discussed.


Functional intermediates in the reaction of membrane-bound cytochrome oxidase with oxygen.

Flash photolysis of the membrane-bound cytochrome oxidase/carbon monoxide compound in the presence of oxygen at low temperatures and in the frozen state leads to the formation of three types of intermediates functional in electron transfer in cytochrome oxidase and reduction of oxygen by cytochrome oxidase. The first category (A) does not involve electron transfer to oxygen between -125 degrees and -105 degrees, and includes oxy compounds which are spectroscopically similar for the completely reduced oxidase (Cu1+alpha3(2+)-O2) or for the ferricyanide-pretreated oxidase (Cu2+alpha3(3+)-O2). Oxygen is readily dissociated from compounds of type A. The second category (B) involves oxidation of the heme and the copper moiety of the reduced oxidase to form a peroxy compound (Cu2+alpha 3(3+)-O2=or Cu2+alpha3(2+)-O2H2) in the temperature range from -105 degrees to -60 degrees. Above -60 degrees, compounds of type B serve as effective electron acceptors from cytochromes a, c, and c1. The third category (C) is formed above -100 degrees from mixed valency states of the oxidase obtained by ferricyanide pretreatment, and may involve higher valency states of the heme iron (Cu2+alpha3(4+)-O2=). These compounds act as electron acceptors for the respiratory chain and as functional intermediates in oxygen reduction. The remarkable features of cytochrome oxidase are its highly dissociable "oxy" compound and its extremely effective electron donor reaction which converts this rapidly to tightly bound reduced oxygen and oxidized oxidase.


Glycoproteins released into the culture medium of differentiating murine neuroblastoma cells.

C-1300 murine neuroblastoma cells release glycoproteins into the culture medium. The process was studied by prelabeling spinner cultures for 12 to 60 hours with [3H]glucosamine. Then, the medium was removed and replaced with fresh medium lacking radioactive isotope. Soluble material released into the medium during the subsequent 2-hour incubation was collected by trichloroacetic acid precipitation. The released proteins were then separated by discontinuous polyacrylamide gel electrophoresis in buffers containing sodium dodecyl sulfate. The electrophoretograms of glycoproteins obtained from cultures labeled for different lengths of time were very similar; three major radioactive regions centered about molecular weights 87,000, 66,000, and 55,000 were present. When spinner cells were transferred to monolayer culture in the presence of N6,O2' dibutyryl adenosine 3':5'-monophosphate (Bt2cAMP), differentiation (extension of neurites twice the diameter of the perikaryon) was observed. Monolayer cultures grown in the presence of Bt2cAMP and [3H]glucosamine for 12 hours released glycoproteins which gave a gel electrophoresis pattern similar to that obtained using spinner cultures. However, after 60 hours in the presence of Bt2cAMP and [3H]glucosamine, the released radioactive material consisted almost exclusively of glycoproteins of the 66,000 molecular weight class. Similar results were obtained if [3H]fucose was substituted for [3H]glucosamine, or if bromodeoxyuridine (which also induced differentiation) was substituted for Bt2cAMP. Similar experiments using radioactive amino acids were conducted with both spinner and monolayer cultures. Much of the released radioactive material was contained in the same three molecular weight classes as the glycoproteins released by spinner cells prelabeled with [3H]glucosamine, and this pattern did not vary with length of labeling period or type of culture. These results may imply that the glycosylation of released proteins is influenced by agents which can induce differentiation. The origin of this released material is discussed. [3H]Glucosamine-labeled glycoproteins of the molecular weight class centered about 55,000 (discussed above) were isolated by preparative gel electrophoresis. They co-migrated with authentic mouse brain microtubular protein as two closely spaced bands on a number of different electrophoretic systems. This protein fraction was also characterized as complexing with a monospecific antitubulin antibody.


High molecular weight RNAs from Rous sarcoma virus and Moloney murine leukemia virus contain two subunits.

The molecular weights of the large genomic RNAs from Rous sarcoma and Moloney murine leukemia viruses were determined by a combination of sedimentation coefficients and retardation coefficients from gel electrophoresis. Six RNA standards, ranging from 0.7 X 10(6) to 5.3 X 10(6) daltons, were employed. Studies in the presence of varying concentrations of Mg2+ showed that the method provided valid molecular weights for RNAs of differing amounts of ordered structure. The molecular weight (X 10(-6)) of the high molecular weight RNA complexe from Rous sarcoma virus was 7.6 (+/-0.3) and from murine leukemia virus was 6.9 (+/-0.3). The molecular weights (X 10 (-6) of their Subunits were 3.3 (+/-0.1) and 2.8 (+/-0.2), respectively. Hence, the large complexes consisted of two, not three or more, subunits plus small associated RNAs. The high molecular weight RNA from cloned Rous sarcoma virus was heterogenous in molecular weight although the apparent molecular radius was constant; stuides were performed on subfractions of the RNA as well as on RNA from virus harvested at various time intervals. The preparations with lowest molecular weight approached a mass equal to twice that of the subunit, with hydrodynamic properties approaching those expected of normal single-stranded RNA.


All intact subunit RNAs from Rous sarcoma virus contain poly (A).

The capacity of RNA subunits of Schmidt-Ruppin Rous sarcoma virus to bind (dT)n was studied by cesium sulfate density gradient centrifugation, sucrose gradient centrifugation, or polyacrylamide gel electrophoresis. Two classes of subunits were observed. In agreement with previous reports, 60 to 70% of the purified subunits were capable of binding (dT)n. The remaining, nonbinding RNAs, although indistinguishable in size from whole subunits, were shown to be fragments that had lost the (rA)n terminus by random nucleolytic cleavage. This was deduced from the molecular weight distribution of the nonbinding RNAs from high molecular weight RNA. In addition, virtually all subunit RNA from early harvest virus, which contains a greater proportion of intact subunit, binds (dT)n.