Mucin-producing carcinomas of the breast: ultrastructural observations. The ultrastructural characteristics of mucin production in mammary mucoid carcinoma, signet ring cell carcinoma (Harris, Wells & Vasudev 1978), papillary carcinoma and lobular carcinoma are compared. The mucin in lobular carcinoma is confined to intracytoplasmic lumina whereas it is present as membrane bound granules in the other three types, although intracytoplasmic lumina also occur in the latter. A possible origin of intracytoplasmic lumina from distended Golgi cisternae is proposed. Possibly, extracellular mucin acts as a mechanical barrier between tumour cells and lymphatics, thus explaining the good prognosis of typical mucoid carcinomas.


Aftercare for psychiatric patients: does it prevent rehospitalization? Focusing on a sample of 421 patients who made from one to 65 visits to an aftercare clinic, the authors replicated and extended and investigation of the effectiveness of psychiatric aftercare for patients discharged from hospital settings. A total of 167 of the patients were rehospitalized at least once during the follow-up period. The findings of both studies suggest that there is a significant relationship between an increasing number of aftercare visits and a reduced liklihood of rehospitalization, especially among the more chronic patients. The authors emphasize the need for further studies in which patients are interviewed directly about their perceptions of aftercare.


Congregate living for the mentally ill: patients as tenants. The authors describe an apartment-living project for chronic mental patients released from the Hillside Division of the Long Island Jewish-Hillside Medical Center. The apartments, which are rented by the hospital and sublet to the patients, are located in modern, well-maintained high-rise buildings within commuting distance from the hospital. To avoid creating a psychiatric ghetto, the project rents no more than two apartments in buildings of a hundred or more units. The hospital was able to rent the apartments by assuring the landlords that the hospital would be a financially responsible tenant and that staff would be in continuing contact with the patients, would be available to the landlords if problems arose, and would remove troublesome tenants. Some of the problems encountered by the patients in the program are described, as are guidelines for selecting those who have a reasonable chance of benefiting from such a program.


A positive look at boarding homes.

A positive look at boarding homes. The authors report on a project in Philadelphia in which boarding homes were successfully used to house former state hospital patients in a warm and therapeutic environment. The program was established jointly by the Jefferson Community Mental Health Center and Horizon House, a psychosocial rehabilitation agency. It drew the proprietors into the treatment program through close contact with professional staff and through meetings and seminars on various aspects of housing and care. The authors note that their positive experiences with boarding homes and their proprietors run counter to the negative experiences reported in much of the literature.


Comparative studies in two cases of testicular feminization syndrome, one with and the other without the fluorescent distal band q12 of the Y. In two patients with testicular feminization syndrome, one Yq12-positive and the other Yq12-negative, we compared phenotype, gonadal histology and ultrastructure, in vivo steroid response of the testes to the administration of exogenous gonadotropin during adrenal suppression, and of the adrenal gland to ACTH. The assumption that the constitutive Y heterochromatin could function as a regulator of steroid biosynthesis and metabolism, with or without effect at other levels, was not firmly supported by the observations reported here. However, further studies along this line may help elucidate the biologic role of this portion of the Y chromosome in humans.


Effect of dibutyryl cAMP on cell cycle progression of rat brain tumor cells in vitro. Autoradiographic and flow microfluorometry analyses have been applied to a study of perturbed cell kinetics in 9L rat brain tumor cells treated with dibutyryl cyclic AMP and theophylline alone and in combination in vitro. At a concentration of 1 mM each, cell growth ceased shortly after the administration of these drugs. The results indicate that cells in S and G2 phase at the time of drug administration can undergo mitosis even though a considerable prolongation of G2 phase was apparent. However, cells in G1 at the time of drug administration was arrested in that phase, whereas those cells in S or G2 were able to complete one mitosis before becoming arrested in the G1 phase. This blocking effect was reversible, and cells resumed proliferation at a normal rate shortly after the removal of these drugs.


A virus-producing cell line developed by transformation of human parapharyngeal cells with SV40. Normal cultures of epithelial appearance were initiated by trypsinization of a surgically resected, histologically normal branchial cyst. Cellular morphology was consistent with derivation from the stratified squamous epithelium of the cyst or from vascular endothelium, although electron micrographs of the cultured cells failed to show any junctional complexes. Infection with SV40 produced transformants which were also epithelioid in appearance. These grew vigorously for 22 to 50 population doublings (about 23 to 32 subcultures, depending upon regimen) and then became quiescent. During this evolution, virus was detectable at all stages by both direct isolation (cell extracts) and cocultivation with permissive cells. In two sublines in which selection for rapidly growing cell types occureed, virus was detected only by cocultivation. The work confirms that of others in the finding that normal human epithelial cells are susceptible to transformation by oncogenic viruses, but are apparently less responsive than are fibroblasts to such transforming agents. It also suggests that subcultivation techniques that maintain the populations of transformed cells at low density tend to select against cell strains that are continous producers of infectious virus.


A transitional cell carcinoma cell line. A transitional cell carcinoma cell line, COLO 232, was derived from a primary urinary bladder tumor in a Caucasian male. In culture, COLO 232 retained distinct uroepithelial phenotypic traits and produced both carcinoembryonic antigen and adrenocorticotropic hormone. COLO 232 had a chromosome mode of 58 and retained the X and Y chromosomes. Ten marker chromosomes were identified. COLO 232 will be of value for biochemical and immunological studies.


Effect of serum on cells cultured from human mammary tumors. Clusters of cells derived from biopsy specimens of human mammary ductal carcinomas form two morphologically distinct epithelial colonies in culture, designated as E and E'. The proportion of E' cell clusters that attached and formed colonies ranged from 0.3 to 13.0% with different tumors. Attachment was independent of tumor grade. Microscopic observations revealed that the survival of E' cell colonies was limited to approximately 10 days with rapid cell degeneration commencing about 7 days. A comparison of sera showed that colony formation by cells from malignant tumors during the 1st week of culture was maximum in the presence of fetal bovine serum. Human serum alone was 70 to 100% less effective in promoting E' colonies. The most significant finding was that human serum from normal donors inhibited E' colony development in the presence of FBS. Although human serum was less effective than FBS in promoting colony formation by clusters of E cells, an inhibition was not observed. Inhibitory activity could not be attributed to either antagonistic hormones or the source of human serum. These results demonstrate that normal human serum contains a factor(s) that exhibits an inhibitory activity specific for human epithelial cells (E') derived from malignant tumors.


Subclones of normal clonal prolactin cells of the rat. We recently established a clone (2B8) of normal rat prolactin cells that secretes only prolactin into the medium. When grown in the presence of thyrotrophin-releasing hormone (TRH), estradiol (E2) or arginine vasotocin (AVT), the cells show increased production of prolactin. Subclones of single cell origin were developed from 2B8 cells exposed for 1 week to TRH, E2 or TRH plus E2. These subclones differ in their response to TRH, E2 or AVT and therefore may possess different receptors for these hormones.


Isolation of function human trophoblast cells and their partial characterization in primary cell culture. Human trophoblast isolation and cell culture procedures were examined to identify variables that enhance secretion of chorionic gonadotropin (HCG) in primary culture. Brief exposure of unminced first-trimester placental specimens to a solution of trypsin-EDTA-DNAse, and isolation of the dispersed cells after Ficoll-hypaque centrifugation yielded primary cultures that were high in HCG secretion and content of epithelial-like cells. The gradual decline in HCG level with time in monolayer culture in these presumptive trophoblast cells was retarded by treatment with theophylline and cyclic adenosine monophosphate. Exposure to methotrexate (MTX) did not increase HCG secretion in normal trophoblast cells, in contrast to a 5-fold stimulation by MTX in the JAR line of choriocarcinoma cells. Clusters of polygonal cells in primary culture progressively lost their capacity to secrete HCG and their epithelial-like morphology. However, they could be maintained as cell strains through approximately 15 passages over a period of 13 to 16 weeks.


Cell-mediated immunity assayed by 51Cr release test in mice infected with mouse adenovirus. Immune spleen cells (ISC) from mice immunized with a sublethal dose of mouse adenovirus (M-Ad) were shown by the 51Cr release test to be cytotoxic to target mouse embryonic cells or lymphoid cells infected with M-Ad. The number of ISC required for release of statistically significant amounts of 51Cr from target cells varied from one sample to another, ranging from 5 to greater than or equal to 30 ISC per target cell. When 24-h-infected mouse embryonic cells were used as targets, the release of 51Cr became evident in 6 h after the addition of ISC to the cells, gradually increased with time, and then leveled off. Cytolytic activity of M-Ad ISC is specific for M-Ad, since ISC do not lyse mouse embryonic cells infected with human adenovirus type 12 and vice versa. Kinetic study of the development of cell-mediated immunity to M-Ad assayed by 51Cr release showed that cytolytic activity of ISC in infected mice became detectable 4 days postinfection, reached its peak level about 7 to 10 days postinfection, and fell to undetectable levels about 10 days thereafter. This is consistent with the data obtained by inhibition of intracellular viral antigen synthesis or by the macrophage migration inhibition test in our prevous reports.


Small-plaque variant of canine herpesvirus with reduced pathogenicity for newborn pups. The plaque characteristics of 13 field isolates of canine herpesvirus were found to be similar. After 312 passages of the F-205 strain in dog kidney cell cultures incubated at 35 degrees C, the virus still was virulent for newborn pups; it also had the macroplaque morphology of the wild-type virus. However, after 20 additional passages at 30 degrees C, a microplaque (mP) variant had emerged as the predominant type. Cloned mP virus retained the small-plaque characteristic after 66 transfers at 30 degrees C. The natural history of the mP strain and some of its biological properties are reported, the most significant being its lack of pathogenicity for newborn pups.


Orofacial herpes simplex virus infection in hairless mice: latent virus in trigeminal ganglia after topical antiviral treatment. Inoculation of herpes simplex virus on the forehead and/or snout of hairless mice resulted in a significantly lower mortality rate than inoculation of the skin in the lumbosacral area. Latent herpes simplex virus infections were detected in all forehead-inoculated and in 90% of snout-inoculated mice. Phosphonoacetic acid was highly effective in preventing the development of skin lesions, and no latent infections were detected when phosphonoacetic acid ointment was applied 3 h after infection. Neither adenine arabinoside nor adenine arabinoside monophosphate prevented the establishment of latent infections in the trigeminal ganglia, although they protected the mice from the fatal outcome of the infection. The antibody response after adenine arabinoside or adenine arabinoside monophosphate treatment was similar to that observed in untreated animals, and it was six to eight times higher than in mice treated with phosphonoacetic acid. Mice without evidence of latent infection had, in general, lower serum antibody titers than those with latent infections in the ganglia. An analysis of the pathogenesis of herpes simplex virus infection in mice treated with adenine arabinoside showed that virus penetration into the nerve endings was delayed and that the amount of free virus in ganglionic homogenates was 10 to 100 times less than that for untreated mice.


Loss on serial passage of rhesus monkey kidney cells of proteolytic activity required for Sendai virus activation. Primary and secondary cultures of rhesus monkey kidney cells supported multiple-cycle replication of Sendai virus, but later passages lost this ability, and this was reflected in decreased plaque formation. Multiple-cycle replication also did not occur in LLC-MK2 cells, a continuous line of RMK cells. Failure of replication in serially passed cells was correlated with a decrease in proteolytic cleavage of a viral surface glycoprotein (Fo), and the ability of cells to support multiple-cycle replication and plaque formation could be restored by the addition of trypsin (0.3 microgram/ml) to the overlay medium. The use of wild-type virus, which requires trypsin, and protease activation mutants that require chymotrypsin or elastase for activation has provided evidence that the activating protease supplied by primary or secondary cells has trypsin-like activity. Inactive virus, with uncleaved Fo glycoprotein, absorbed to primary or secondary cells but did not infect them, even though such cells possess the enzyme that is capable of cleaving the Fo glycoprotein of virus synthesized in these cells. The inability of these cells to activate adsorbed virus indicates that the activating protease that they possess is inacessible to adsorbed virus, although it can act on the Fo glycoprotein during virus maturation in these cells. These data provide a biochemical explanation for the failure of later passages of a cell strain or a continuous cell line to support the replication of a paramyxovirus.


Direct syncytial assay for the quantitation of bovine leukemia virus. A direct in vitro tissue culture method is described for the quantitation of bovine leukemia virus, utlizing a feline S+L-- cell line.


Effect of Mycobacterium tuberculosis BCG infection on the resistance of mice to ectromelia virus infection: participation of interferon in enhanced resistance. Mice infected with Mycobacterium tuberculosis BCG were more resistant than normal mice to ectromelia virus infection. It is suggested that enhanced interferon production in peritoneal exudate cells and spleen cells of BCG-infected mice plays an important role in this resistance.


In vitro establishment of Marek's disease herpesvirus-transformed productive and nonproductive lymphoblastoid cell lines. The establishment of two Marek's disease herpesvirus-transformed lymphoblastoid cell lines (JMV-1 and JM-1) is reported. Cocultivation with chicken embryo fibroblasts, attempts to rescue Marek's disease virus from kidney cell cultures, and immunofluorescent staining show that JMV-1 is a nonproductive line and JM-1 is a productive line.


Bluetongue virus, an exceptionally potent interferon inducer in mice. The attenuated American BT-8 strain of bluetongue virus is 5 to 10 times more potent an interferon inducer than any other viral or nonviral agent reported to date, including as much as 600,000 units/ml of plasma by 8 h after intravenous injection.


Effect of zinc and calcium ions on the production of alpha-toxin and proteases by Clostridium perfringens. Clostridium perfringens produced at least three distinct proteases in a synthetic medium containing calcium. Two of them, thiol and ethylenediaminetetraacetic acid disodium salt-sensitive proteases, appeared at an early stage of growth, but the other one, perhaps being identical to the one produced in a calcium-deficient medium, appeared at a late stage. The production of these proteases depended on Ca2+ but not on Zn2+ in the medium. Alpha-toxin, perhaps being a zinc-containing metalloenzyme, was rather resistant to the proteases, but toxin, produced in a zinc-deficient medium or deprived of zinc with ethylenediaminetetraacetic acid disodium salt, was very sensitive. By adding Zn2+, the toxin lacking zinc may have been converted to the zinc-containing metalloprotein that is resistant to proteases. This may explain why alpha-toxin activity increased progressively in a zinc-containing medium in spite of simultaneous production of potent proteases and why it disappeared rapidly in a zinc-deficient medium.


Properties of an Escherichia coli cytotoxin. Isoelectric focusing of a heat-labile cytotoxin of Escherichia coli H30 revealed the presence of two molecular variants, pI 7.2 and a comparatively small quantity of pI 6.8. Predominant component pI 7.2 had a molecular weight of 28,000, induced some fluid accumulation in rabbit ileal loops, and showed no morphological response in Y-1 cells but a strong cytotoxic effect on Vero cells.


Innate cytotoxicity of CBA mouse spleen cells to Sendai virus-infected L cells. The presence in the spleens of unsensitized CBA mice of cells that are spontaneously cytotoxic for Sendai virus-infected L cells was confirmed. This innate cytotoxic activity to virus-infected cells was shown to exhibit some H-2 restriction. Partial identity of only the D end of the H-2 gene complex between the target and effector cells was required to produce cytolysis. Attempts to characterize the kind of cell active in this system indicated that neither the theta antigen nor the surface immunoglobulin markers were present. Furthermore, the cells appeared to have no adherent or phagocytic properties. The relationship between the effector cells responsible for innate cytotoxicity to virus-infected cells and the natural killer (NK) cells spontaneously cytotoxic for certain tumor cells is discussed.


Proliferative and interferon responses by peripheral blood mononuclear cells after bone marrow transplantation in humans. The capacity of peripheral blood mononuclear cells from bone marrow transplant recipients to proliferate and produce interferon in response to mitogens and specific antigens was tested. Proliferation in response to phytohemagglutinin or pokeweed mitogen occurred in cells from more than 90% of the recipients, and interferon was present in 60 to 70% of the supernatants from these cultures, even when tested as soon as 8 weeks after transplantation. Proliferation in response to bacterial antigens was infrequent, and interferon release was not detected. In the early post-transplantation period (less than 13 weeks), cells from only two of four cytomegalovirus (CMV) antibody-positive patients proliferated normally in response to CMV antigen and interferon release was detected only once. In the late post-transplantation period (more than 13 weeks), in only two of five instances did cells proliferating in response to CMV antigen release interferon. The response to CMV antigen of mononuclear cells from many transplant recipients differs from that of cells from normal controls.


Replication of dengue and junin viruses in cultured rabbit and human endothelial cells. The flavivirus dengue and the arenavirus Junin are both associated with a hemorrhagic shock syndrome in man. We have demonstrated the replication of these viruses in vitro in both rabbit and human endothelial cells by viral titers and immunofluorescent antibody studies. Rabbit endothelium established in continuous culture was derived from vena cava, while human cells in primary culture were derived from umbilical veins. In rabbit endothelium, dengue-2 virus passaged through monkey kidney monolayer cells (LLC-MK2) or human lymphoblastoid cells (raji) produced significantly more virus than the seed obtained from suckling mouse brain (MB). Inoculation of actively dividing, subconfluent human endothelial cells with the LLC-MK2 degue virus produced higher viral titers than inoculation of confluent cells. The appearance of Junin virus was delayed beyond that of dengue virus in rabbit endothelial cells although equivalent titers of virus were produced. In human cells, Junin virus was less productive than dengue virus and produced characteristic cycles of virus release. This is the first direct evidence for replication of human hemorrhagic fever viruses in endothelial cells.


Characterization of the TO strains of Theiler's mouse encephalomyelitis viruses. Theiler's mouse encephalomyelitis virus isolates from the central nervous systems of spontaneously paralyzed mice and stools of asymptomatic mice resemble Theiler's original virus isolates. In this study four such strains were adapted by blind subpassage to replicate and to produce cytopathic effect in cell culture. These viruses were then found to be closely related to each other and to GDVII virus by cross-neutralization and to form small plaques. Bovine serum was found to contain cross-reacting antibodies to these viruses.


Mouse natural killer (NK) cell activity against human cell lines is not influenced by superinfection of the target cell with xenotropic murine C-type virus. Mouse natural killer (NK) cells can lyse a variety of syngeneic, allogeneic and xenogeneic target cells in short-term 51Cr release assays. The target specificity of NK cells is not known, but endogenous C-type viral antigens have been suggested as possible target structures. To test this hypothesis, human lymphoid lines were superinfected with xenotropic mouse C-type virus either by repeated dosage through nude mice or by in vitro superinfection with the supernatants of nude-mouse-passaged lines. The appearance of surface-associated MuLV antigens after superinfection was confirmed in a complement-dependent cytotoxicity test. Subsequently, the NK sensitivity of each infected line was compared with its non-infected counterpart in direct cytolytic and competition assays. None of these two assay systems showed a consistent difference in NK sensitivity of infected and non-infected cell lines. These findings do not lend support to the concept tht murine C-type viral antigens are responsible for NK sensitivity.


Differential growth and transmission in cats of feline leukaemia viruses of subgroups A and B. The outcome of infecting cats with FeLV of sub-groups A (FeLV-A) or B (FeLV-B) was different. After FeLV-A infection, virus was quickly recovered from the blood and oropharynx of most animals. Following infection with FeLV-B, only a small proportion of cats developed a viraemia, and this after a long interval. There was no evidence that FeLV-B was transmitted by contact. The result of infecting cats with mixtures of FeLV-A and B (FeLV-AB) was that to a large extent each virus operated independently. FeLV-A was recovered from the plasma first, in a high proportion of the cats, and FeLV-B appeared later but not in all cats. There was evidence of interaction, however, in that the proportion of cats which were viraemic with FeLV-B was greater following FeLV-AB infection than after infection with FeLV-B alone; also FeLV-B was transmitted by contact from cats excreting FeLV-AB. These results help to explain the apparent dependence of FeLV-B on FeLV-A under natural conditions and the frequency of occurrence of sugroups A and B in FeLV isolates.


Genetic transmission of mammary tumour virus in the DBAf mouse strain. MTV antigens were demonstrable by radioimmunoassay in milk samples from individual DBAf mice, and in samples from (male BALB/c X female DBAf) F1 mice. Although some samples collected during the first lactation periods of these mice were virus-negative, all samples of later lactation periods were virus-positive. From 75 mice of the [ male BALB/c X female (male BALB/c X female DBAf)]Bc I population, milk samples were collected during one or more lactation periods and tested for the presence of viral antigens; the samples of 42 mice were virus-positive. In the ([BALB/c X (BALB/c X DBAf)] X BALB/c)Bc II population two groups were distinguished. In the first group, the progeny of virus-positive Bc I mothers, 37 out of 62 mice had detectable levels of viral antigen in the milk. None of the 43 samples from mice of the second group, derived from MTV-negative Bc I females, were virus-positive. These data suggest that the presence of viral antigens in the milk of DBAf mice is controlled by a single dominant gene; evidence for linkage of this gene and the albino locus was obtained (recombination percentage: 20).


Circulating immune complexes in rats bearing chemically induced tumors. I. Sequential determination during the growth of tumours at various body sites. Circulating immune complexes were measured in sequential monitoring studies in rats bearing chemically induced tumours at different body sites. The assay employed were based upon radioimmunoprecipitation of serum factors with 125I-C1q or with an indirect test whereby the inhibition of binding of 125I-C1q to IgG aggregates by tumour-bearer sera was measured. 125I-C1q-binding immune complexes were detected at the initial phase of tumour growth and the level of this activity returned to the normal range after tumour excision. With tumour growing at subcutaneous sites or in th peritoneal cavity, serum-borne C1q-binding material, after reaching a maximum level, decreased despite tumour progression and eventually fell into sub-normal ranges at terminal stages of growth. In contrast, following intravenous tumour cell injection, immune-complex levels increased until terminal stages of disease. The present findings indicate that the measurement of C1q-binding serum factors represents a useful method for monitoring the growth and burden of experimental animal tumours.


Neoplastic transformation of hamster brain cells in vitro by polyoma virus. The transformation of cultivated hamster brain cells by polyoma virus is reported. The transformed cell line contained polyoma virus-specific nuclear, surface and transplantation antigens. Subcutaneous and intracranial inoculations revealed high tumorigenicity of the cells. Brain-specific S 100 protein was found in these tumors with immuno-peroxidase staining, suggesting that they were of a nervous nature. Both in vivo and in vitro, the cells had glial features as studied by phase contrast, light and electron microscopy. Type-H virus-like particles were found in the tumor cells and might have played a role in the viral transformation.


Search for infective mammalian type-C virus-related genes in the DNA of human sarcomas and leukemias. DNA was extracted from two human sarcoma cell lines, TE-32 and TE-418, and the leukemic cells from five children with acute myelocytic leukemia, three children with acute lymphocytic leukemia and four adults with acute myelocytic leukemia. The DNAs, assayed for infectivity by transfection techniques, induced no measurable virus by methods which would detect known mammalian C-type antigens or RNA-directed DNA polymerase in TE-32, D-17 dog cells and other indicator cells, nor did they recombine with or rescue endogenous human or exogenous murine or baboon type-C virus. Model systems used as controls were human sarcoma cells, TE-32 and HT-1080, and human lymphoma cells TE-543, experimentally infected with KiMuLV, GaLV or baboon type-C virus, all of which released infectious virus and whose DNAs were infectious for TE-32 and D-17 dog cells. Other model systems included two baboon placentas and one embryonic cell strain spontaneously releasing infectious endogenous baboon virus and yielding DNAs infectious for D-17 dog cells but not for TE-32 cells. Four other baboon embryonic tissues and two embryonic cell strains, releasing either low levels of virus or no virus, did not yield infectious DNA.


Activation of endogenous type-C viral p30 antigen in chemically-induced rat hepatocellular carcinomas. Chemically induced rat hepatocellular carcinomas were prospectively examined for expression of the major group-specific antigen (p 30) of genetically transmitted rat type-C viruses. Ten primary tumors induced following oral administration of the carcinogen N-2-acetylaminofluorine did not express elevated levels of viral antigen as compared to antigen levels detected in normal liver tissue. By contrast, rapidly growing transplantable hepatocellular carcinomas (THCs) derived from primary tumor expressed increased quantities of viral antigen. The expression of antigen was marginally elevated after only one transplanation, increased to maximal levels after several transplant generations and, once achieved, was stably maintained throughout subsequent transplants. Studies with additional previously established THCs showed that poorly differentiated, rapidly proliferating tumors tended to express elevated levels of viral antigen, while more differentiated, slowly growing tumors did not. The results show that the expression of endogenous type-C viral 30 antigen is a stable phenotypic property of many rat THC lines.


Further characterization of a herpesvirus-positive orang-utan cell line and comparative aspects of in vitro transformation with lymphotropic old world primate herpesviruses. An orang-utan (Pongo pygmaeus) suspension line, CP81, was shown to lack myeloid markers of lysozyme activity an d phagocytosis but to be positive for lymphocytic N-alkaline phosphatase activity, and to release a B-cell-tropic herpesvirus. This herpesvirus, termed Herpesvirus pongo, had 30--40% DNA homology with EBV and was present at 2-3 genome copies per CP-81 cell. Gibbon lymphocytes transformed by H. pongo, Epstein-Barr virus (EBV), and H. papio (of baboon, Papio hamadryas, origin) were found to be virus antigen-positive B cells. Gibbon lymphocytes transformed by H. pongo and EBV and transplanted to nude mice by the intracranial (IC) route (had a 75% and a 45% success rate, respectively), while transplants of similar cells transformed by H. papio were only 10% successful. None of these lines transplanted subcutaneously (SC) nor manifested a high degree of colony formation in 0.33% agarose (less than or equal to 0.5%), Gibbon lymphocytes transformed by H. pongo were hypodiploid while those transformed by EBV or H. papio were diploid. CP-81 cells themselves could be transplanted both IC (100%) and SC (70%) and showed a relatively high degree of colony formation in agarose (6.4-7.6%). B95-8 cells (marmoset, Saguinus oedipus-EBV) could be transplanted IC (66%) but not SC and had a low but significant ability to grow in agarose (1.6%). 594S (baboon, P. hamadryas-H. papio) cells could be transplanted IC (25%) but not SC, and grew to very low levels in agarose (0.1%).


The specificity of neutralizing antibodies to feline leukaemia viruses. Feline leukaemia viruses (FeLV) of subgroups A (FeLV-A), B(FeLV-B) and C(FeLV-C) were examined by neutralization. The FeLV-A isolates were monotypic. By contrast, there was antigenic variation within subgroups B and C. There was also considerable cross-reactivity between subgroups. Thus, FeLV-A viruses showed weak cross-reactivity to the standard FeLV-C isolate; four of the subgroup B viruses were identical and cross-reactied with the standard FeLV-C, but not with FeLV-A or the fifth FeLV-B, and this latter virus weakly cros-reacted with FeLV-A but not with the FeLV-C; within subgroup C, one virus was indistinguishable from the standard FeLV-C strain but the other two were very similar to FeLV-A. Therefore, subgroup classification based on interference did not altogether correspond with neutralizing behaviour for subgroups B and C.


Reaching problem-drinking Blacks: the unheralded potential of drinking driver programs. The potential of court-enforced treatment of problem drinking drivers in reaching and effectively treating problem drinking Blacks is examined through an analysis of the experience of one such drinking driver program. The findings support the contention that the potential of court-enforced treatment is significant and should not be overlooked in any plan aimed at providing treatment services to the Black community.


Demonstration of two different types of beta1-receptors in man (selective blockade of the positively inotropic and the positively chronotropic effect of isoproterenol). The hemodynamic effect of a beta-receptor blockade with mepindolol--a noncardioselective beta-receptor blocker--was studied in 6 male test subjects age 25 to 30 years with an increasing dose of isoproterenol. The cardiac output per min, the heart rate and the stroke volume, the wet blood pressure and the contractility parameters dp/dt max in the right and left ventricles were measured as part of the study. It was found that a dissociated right shift of the dose-effect curves occurred for the stroke volume, contractility parameters and heart rate. The following major conclusions can be made from an exact analysis of this result: 1. Evidence was produced that a distinction must be made in man with respect to the so-called beta1-receptors between those which mediate a specific effect on the heart rate and those which mediate a primarily positively inotropic effect. 2. There are apparently some beta-receptor blockers which inhibit the frequency receptors primarily and the inotropic receptors to a lesser extent. 3. Through their beta 2-effect, noncardioselective beta-receptor blockers can partly compensate for their negatively inotropic effect on the heart by maintaining the Frank Starling mechanism.


Studies on the retina and the pigment epithelium in hereditary canine ceroid lipofuscinosis. II. The subcellular distribution of lysosomal hydrolases and other enzymes. Observations on the progressive course of retinal degeneration in canine ceroid lipofuscinosis (CCL) showed dramatic changes in enzyme activity and subcellular compartmentation. Thus, in affected animals, a new particle containing high levels of hexosaminidase and galactosidase was found in fractions lighter than seen in controls. In the later stages of disease and in normal aging, a progressive increase in dense fractions with high titers of acid lipase and acid phosphatase was observed. Peroxidase was found predominantly in the heavier fractions (1.24 to 1.28 gm/ml) and was lower than normal in affected retina and RPE. These fractions were located above the pigment granule fraction. Changes of peroxidase activity in the pigment granules were age dependent in controls, but a decrease of similar magnitude occurred much earlier in affected dogs. The accumulation of large numbers of dense bodies in the retina and RPE in CCL may indicate an impairment of intracellular digestive mechanisms. The early and marked reduction of peroxidase activity in affected dogs is an important indicator for major changes in the biochemistry of the entire eye in this disease. Therefore the initial pathogenic event seems to be the inability of affected cells to cope with peroxidative damage at an early stage, followed by an exaggerated attempt by the cells to digest accumulating lipopigments.


Association of cytomegalovirus (CMV) infection with cervical cancer: isolation of CMV from cell cultures derived from cervical biopsy. Cytomegalovirus (CMV) was isolated from cell cultures derived from 2 of 10 cervical cancer biopsies from patients in an advanced stage of the disease. Five serial passages were necessary before extensive cytopathic changes characteristic of CMV infection appeared. All patients tested had complement-fixing antibodies to both isolates in higher titers than to the prototype AD169 strain. 6 of 8 patients tested also had herpesvirus type 2 antibodies.


Tumor induction in newborn and adult Syrian hamsters infected with SV40 temperature-sensitive mutants. Newborn and adult Syrian hamsters were injected with wild-type SV40 and its temperature-sensitive (ts) mutants A30, A209, A239, B201 and BC210. Wild-type SV40 and ts B201 were highly oncogenic for newborn but not adult animals; ts A239, ts A30 and ts BC210 also induced tumors in some adult hamsters. Unexpectedly, the number of tumors induced by ts A209 and ts A239 in newborn animals was very low. The number of tumors observed was also low in adults infected with ts A30, ts A239 and ts BC210. The duration of the latent period of tumors induced in adult animals was the same as in the newborns. The data obtained are discussed in connection with the defectiveness of the mutants to induce tumor-specific transplantation antigen activity in hamster cells.


Inhibition of DNA metabolism in human B lymphocytes by a substrain of Epstein-Barr virus (P3HR-1): a method for virus quantitation. Pretreatment with the P3HR-1 substrain of Epstein-Barr virus (EBV) inhibited approximately 85% of the DNA stimulation induced by Staphylococcus aureus in human B lymphocytes. In parallel experiments, the DNA stimulation induced by the transforming B95-8 substrain of EBV was almost completely inhibited by prior exposure to P3HR-1 virus. Phytohemagglutinin stimulation was only slightly inhibited, whereas the DNA synthesis of continuously growing Raji cells was inhibited to a considerable extent. The degree of DNA inhibition was correlated with the induction of the EBV-determined antigens (membrane, early, and nuclear antigens) in sensitive indicator cells.


Mouse hepatitis virus strain MHV-S: formation of pseudotypes with a murine leukemia virus envelope. Mouse hepatitis virus strain MHV-S forms pseudotype virions enveloped with Friend leukemia virus material when propagated in SRCDF1 DBT cells persistently infected with Friend leukemia virus.


Polyploidization of Epstein-Barr virus (EBV)-carrying lymphoma lines decreases the inducibility of EBV-determined early antigen following P3HR-1 virus superinfection or iododeoxyuridine treatment. Four tetraploid cell lines were derived from the Epstein-Barr virus (EBV)-carrying, nonproducer Burkitt lymphoma line Raji after colcemide treatment. EBV early antigen (EA) was induced by P3HR-1 virus superinfection and by iododeoxyuridine treatment. Inducibility was reduced in all tetraploid lines compared to the near-diploid counterpart. The average number of EBV genome equivalents per tetraploid cell was 60-70, compared to 50 in diploid cells. This contrasts with the EBV genome amplification previously observed in somatic cell hybrids.


Isobaric inert gas supersaturation: observations, theory, and predictions. An isobaric inert gas supersaturation model incorporating both diffusion and perfusion properties of biological tissue is presented in a form which allows ready comparison with experimental observations. This model requires only measurement of inert gas flux and blood gas solubility in order to evaluate "counterdiffusion potential". Inert gas flux across the skin of Yorkshire piglets anesthetized with pentobarbital was measured for He, Ne, CH4, C2H4, N2O, and SF6. Model predictions based upon these data compare favorably with published reports of isobaric inert gas supersaturation, as well as several previously unpublished observations. The possibility of supersaturation resulting from the use of hydrogen as a breathing gas in a helium environment is also discussed, and extensive animal testing is recommended before potentially dangerous human exposure occurs.


Primary role of respiratory afferents in sustaining breathing rhythm. We studied the effects on breathing rhythm of suppressing the major respiratory stimuli (wakefulness, vagal, peripheral and central chemoreceptors) in healthy, unanesthetized dogs. Respiratory frequency (f) was obtained with a pneumotachograph; the state of wakefulness (W) or sleep was determined by EEG and behavioral criteria. During quiet W, f averaged 17 breaths/min and minute volume of ventilation (VI), 8.4 l/min. In slow-wave sleep (SWS), f slowed to 14 breaths/min, and VI decreased to 6.8 l/min. Afferent vagal blockade during SWS slowed f to 4 breaths/min, due primarily to prolongation of expiratory duration (Te) to 13.3 s, and decreased VI to 4.8 l/min. One breath of 100% O2 prolonged Te further to 27.4 s. Central chemoreceptor sensitivity was then reduced by inducting a metabolic alkalosis that combined with SWS, vagal blockade, and hyperoxia prolonged Te to as long as 57 s and reduced f to as low as 1 breath/min. The results demonstrate that afferent respiratory stimuli are essential for sustaining adequate ventilation.


Cyclic phosphates of formycin.

Cyclic phosphates of formycin. The syntheses of N1- and N2-isopropylformycin (10, 11), formycin 3',5'-cyclic and 2',3'-cyclic phosphate (3,7) and their N-methyl and N-isopropyl derivatives (13, 15, 19, 23) are described. It was observed that substitution at N1 or N2 with a bulky alkyl group or cyclic phosphorylation of the ribose moiety made formycin resistant to adenosine deaminase.


Cell-free synthesis of proteins related to sn-glycerol-3-phosphate transport in Escherichia coli. An Escherichia coli periplasmic protein (GlpT) related to sn-glycerol-3-phosphate transport was synthesized in a cell-free system directed by hybrid plasmic ColE1-glpT DNA. The in vitro product cross-reacted with antisera against the purified protein. The ColE1-glpT DNA-directed cell-free system was induced by sn-glycerol-3-phosphate and phosphonomycin and was dependent on cyclic AMP. The in vitro-synthesized protein showed the characteristics of a multimeric protein, as did the purified periplasmic protein. The main proportion of the newly synthesized product had a higher molecular weight than the mature protein found in the periplasm of cells and showed a more positive charge in two-dimensional gel electrophoresis. Thus, a proportion of this protein is presumed to be synthesized in vitro as a precursor. The cell-free system yielded a second protein that is likely to be also coded for by the glpT operon. This protein had a molecular weight of approximately 33,000 in sodium dodecyl sulfate-acrylamide gel electrophoresis and behaved like an intrinsic membrane protein.


Kinetics of glucose repression of yeast cytochrome c. The kinetics of glucose repression of cytochrome c synthesis was measured by a radioimmune assay. When 5 or 10% glucose was added to a derepressed culture, the rate of cytochrome c synthesis was reduced to the repressed level with a half-life of 2 min. The addition of 1 or 0.5% glucose repressed the rate of cytochrome c synthesis to the same level as high glucose concentrations but with a longer half-life of 3 min. Glucose repression had no effect on the stability or function of the cytochrome c protein. Cellular levels of active cytochrome c mRNA during glucose repression were measured by translation of total cellular polyadenylic acid-containing RNA and immunoprecipitation cytochrome c from the translation products. The results of these measurements indicate that glucose represses the rate of cytochrome c synthesis through a reduction in the level of translatable cytochrome c mRNA.


Studies on pig serum lipoproteins. V. Optical properties of low density lipoproteins. The circular dichroism (CD), optical rotatory dispersion (ORD), and fluorescence emission spectra of two subfractions of pig serum low density lipoproteins (LDL1 and LDL2) were compared. The contribution of the carbohydrate moiety to the CD and ORD spectra was estimated on the basis of data obtained from isolated glycopeptides and the constituent monosaccharides. The carbohydrate moiety had no effect on the conformation of the protein moieties of LDL1 and LDL2 (apoLDL1 and apoLDL2). However, the intensities of the observed extrema in the CD and ORD spectra of the glycopeptides were greater than those expected from the monosaccharide composition. This suggests the existence of secondary structure in the carbohydrate moiety. In contrast to the carbohydrate moiety, the contribution of the lipid moiety to the CD and ORD spectra could not be neglected. When the effect of the lipid moiety was subtrated from the CD and ORD spectra, the spectra due to apoLDL1 and apoLDL2 were quite similar. Delipidation in the presence of sodium dodecyl sulfate (SDS) induced an increase in the content of disordered structure and alpha-helix accompanied by a decrease in the beta-structure. In the presence of SDS, marked quenching occurred in the fluorescence emission spectra with a blue shift of the maximum emission wavelength from 332 to 326 nm. ApoLDL1 and apoLDL2 showed quite similar SDS-induced conformational transitions. The secondary structures of apoLDL1 and apoLDL2 in the native lipoproteins were stable to changes of pH and temperature. However, this stability was lost in the presence of SDS. These results suggest the importance of the lipid moiety in maintaining the native secondary structures of LDL1 and LDL2. From the overall similarity of the optical properties of apoLDL1 and apoLDL2, we conclude that the secondary structures of apoLDL1 and apoLDL2 are identical.


The serine protease from rat liver and hepatoma 8999. Location and role in mitochondrial protein degradation. 1. Hepatoma 8999 showed extremely high activity of serine protease, but similar activities of other lysosomal proteases to those of normal rat liver. 2. Serine protease from rat liver formed a single immunoprecipitation band against antiserum to purified protease from hepatoma 8999. 3. The serine proteases in rat liver and hepatoma 8999 were restricted to the inner membranes of the mitochondrial fraction. 4. Polyacrylamide gel electrophoresis with sodium dodecylsulfate showed that hepatoma 8999 mitochondria contained less of the slowest moving protein component than rat liver mitochondrial protein. This component was found to be the best substrate for mitochondrial serine protease in both liver and hepatoma 8999. 5. The role of serine protease in mitochondrial protein degradation is discussed on the basis of these results.


A new extragenic suppressor of cya mutation. Mutant cyclic AMP receptor protein with an increased affinity for cyclic AMP. A strain bearing an extragenic suppressor of cya mutation was isolated as a second-site revertant of an adenylate cyclase deficient strain. The mutant was unable to synthesize cAMP but showed normal fermentation profiles and growth properties on a variety of carbon sources. The site of reversion was mapped in, or near, the structural gene for the cAMP receptor protein. Structural alteration of the protein was directly demonstrated by the following biochemical observations: (i) A 10-fold decrease in the dissociation constant for cAMP, (ii) an acidic shift in the isoelectric point, and (iii) the altered binding properties to lambdah80dlac ps DNA.


Calcium sensitivity of contractile proteins from chicken gizzard muscle. Actin, myosin, and "native" tropomyosin (NTM) were separately isolated from chicken gizzard muscle and rabbit skeletal muscle. With various combinations of the isolated contractile proteins, Mg-ATPase activity and superprecipitation activity were measured. It was thus found that gizzard myosin and gizzard NTM behaved differently from skeletal myosin and skeletal NTM, whereas gizzard actin functioned in the same wasy as skeletal actin. It was also found that gizzard myosin preparations were often Ca-sensitive, that is, that the two activities of gizzard myosin plus actin without NTM were activated by low concentrations of Ca2+. The Mg-ATPase activity of a Ca-insensitive preparation of gizzard myosin was not activated by actin even in the presence of Ca2+. When Ca-sensitive gizzard myosin was incubated with ATP (and Mg2+) in the presence of Ca2+, a light-chain component of gizzard myosin was phosphorylated. The light-chain phosphorylation also occurred when Ca-insensitive myosin was incubated with gizzard NTM and ATP (plus Mg2+) in the presence of Ca2+. In either case, the light-chain phosphorylation required Ca2+. Phosphorylated gizzard myosin in combination with actin was able to exhibit superprecipitation, and Mg-ATPase of the phosphorylated gizzard myosin was activated by actin; the actin activation and superprecipitation were found to occur even in the absence of Ca2+ and NTM or tropomyosin. The phosphorylated light-chain component was found to be dephosphorylated by a partially purified preparation of gizzard myosin light-chain phosphatase. Gizzard myosin thus dephosphorylated behaved exactly like untreated Ca-insensitive gizzard myosin; in combination with actin, it did not superprecipitate either in the presence of Ca2+ or in its absence, but did superprecipitated in the presence of NTM and Ca2+. Ca-activated hydrolysis of ATP catalyzed by gizzard myosin B proceeded at a reduced rate after removal of Ca2+ (by adding EGTA), whereas that catalyzed by a combination of actin, gizzard myosin, and gizzard NTM proceeded at the same rate even after removal of Ca2+. However, addition of a partially purified preparation of gizzard myosin light-chain phosphatase was found to make the recombined system behave like myosin B. Based on these findings, it appears that myosin light-chain kinase and myosin light-chain phosphatase can function as regulatory proteins for contraction and relaxation, respectively, of gizzard muscle.


Studies on pig serum lipoproteins. VI. Surface charge of very low density lipoprotein. The surface electric charge of pig serum very low density lipoprotein (VLDL) is described. By isoelectric focusing VLDL was separated into at least 3 fractions having different isoelectric points and polypeptide distributions. The ultracentrifugal and electron microscopic results indicate that the VLDL was not drastically denatured by Ampholine.


Muscarinic cholinergic receptor modulation of beta-adrenergic receptor affinity for catecholamines. The effects of the muscarinic cholinergic agonist methacholine on affinity of beta-adrenergic receptors for isoproterenol and on isoproterenol-induced stimulation of adenylate cyclase activity were assessed in canine myocardium. GTP and guanyl-5'-yl imidoiphosphate both decreased the affinity of beta-adrenergic receptors for isoproterenol without altering the affinity of these receptors for propranolol. Methacholine (10 nM to 10 micronM) antagonized the guanine nucleotide-induced reduction in beta-adrenergic receptor affinity for isoproterenol. This effect of methacholine was reversed by atropine. The choline ester had no effect on the affinity of beta-adrenergic receptors for isoproterenol in the absence of guanine nucleotides. Likewise, methacholine had no effect on the affinity of beta-adrenergic receptors for propranolol, either in the presence or absence of guanine nucleotides. Methacholine also attenuated GTP-induced activation of adenylate cyclase or isoproterenol-induced activation of the enzyme in the presence of GTP. The effects of methacholine on myocardial adenylate cyclase activity were apparent only in the presence of GTP. These effects were also reversed by atropine. The choline ester had no effect on adenylate cyclase activity in the presence of guanyl-5'-yl imidodiphosphate or NaF. The results of the present study suggest that muscarinic cholinergic agonists can regulate both beta-adrenergic receptors and adenylate cyclase by modulating the effects of GTP.


Gradients of O2 concentration in hepatocytes. 1. Cytochrome P/450-dependent mixed function oxidations of hexobarbital, phenyramidol, and alprenolol in intact hepatocytes were examined at different steady state oxygen concentrations. Apparent Kmo2 values were determined to be 6.4 +/- 1.7, 3.6 +/- 0.6, and 9.8 +/- 1.2 micronM, respectively. 2. Apparent Kmo2 values for metabolism of hexobarbital and alprenolol by liver microsomes were 4.3 +/- 0.4 and 8.7 +/- 0.7 micronM, similar to the corresponding values for whole cells. Therefore, no detectable gradient of O2 concentration exists between extracellular space and endoplasmic reticulum of hepatocytes at these oxygen concentrations. 3. Steady state concentrations of ATP, ADP, AMP, lactate, and pyruvate at different steady state oxygen concentrations were used as indicators of mitochondrial oxygen dependence in intact hepatocytes. Half-maximal changes occurred at [O2] = 12.6 micronM for cytoplasmic [NAD+]/[NADH] (estimated from [lactate]/[pyruvate]), at 7.0 micronM for [ATP]/[ADP], and at 2.8 micronM for adenylate energy charge. The apparent cellular respiratory Kmo2 was 1.90 +/- 0.18 micronM. 4. Comparison of values for oxygen dependence of mitochondrial functions in isolated hepatocytes with published values for isolated mitochondria suggests that a substantial intracellular oxygen gradient exists between the outer cellular membrane and the mitochondrial inner membrane at po2 values below the critical O2 tensions.


Structural determination and stereospecificity of the choleragen-catalyzed reaction of NAD+ with guanidines. The products of the choleragen-catalyzed reaction of NAD+ with guanidine HCl or L-arginine have been isolated by high performance liquid chromatography. Analysis of 1H NMR spectra obtained at 360 MHz establishes the structure of the isolated products to be adenosine diphosphoribosyl guanidiniums present as 1:1 mixtures of alpha and beta anomeric forms. Direct observation of the choleragen-catalyzed reaction of NAD+ with L-arginine by 1H NMR spectroscopy establishes that choleragen synthesizes an alpha anomeric linkage that is subject to subsequent anomerization. The stereospecificity of the reaction provides further evidence that choleragen behaves as an enzyme and may be related mechanistically to other ADP-ribosyltransferases.


Isolation of subfractions of human very low density lipoproteins by zonal ultracentrifugation. Very low density lipoproteins (VLDL) have been isolated and subfractionated on the basis of their differing flotation rates. The procedure consists of a single 45-min zonal ultracentrifugation step using a linear density gradient of d = 1.00 to 1.15 g/ml. Appropriate fractions of the zonal rotor effluent containing the entire VLDL spectrum were characterized by analytical ultracentrifugation, gel filtration chromatography, and complete chemical analysis. Flotation rates of VLDL subspecies from hypertriglyceridemic and normolipemic plasmas correlated directly with their Stokes radii and triglyceride content and inversely with their proportion of cholesterol, cholesteryl esters, phospholipids, and total protein. There was also an inverse correlation of flotation rate with the fraction of tetramethylurea-insoluble protein. This procedure provides a reliable methodology for a rapid isolation of VLDL subfractions and the accurate determination of their flotation rates.


The role of phosphatidylglycerol in the activation of CTP:phosphocholine cytidylyltransferase from rat lung. The reaction catalyzed by CTP:phosphocholine cytidylyltransferase in the reverse direction, i.e. the formation of CTP and phosphocholine from CDP-choline and pyrophosphate, is slightly faster than the reaction in the forward direction. The reverse reaction is optimal at 2 mM pyrophosphate and 6 mM Mg2+, in both fetal and adult preparations. The apparent substrate Km values for phosphocholine, CDP-choline, and pyrophosphate are similar in the fetal and adult forms of the enzyme. The enzyme activity is separated into two forms by gel filtration. The enzyme from adult lung exists as a high molecular weight species, ranging in size from 5 X 10(6) to 50 X 10(6). The enzyme from fetal lung exists as a 190,000 molecular weight species and is totally dependent upon added anionic phospholipid for activity in both the forward and reverse direction. The addition of phosphatidylglycerol gives maximal activity, while phosphatidylinositol or cardiolipin produce about 60 to 70% of the maximal activity. Enzyme activation is accompanied by an aggregation of the enzyme. A sonicated preparation of phosphatidylglycerol is a more efficient activator than a preparation mixed on a Vortex mixer (KA = 30 micronM) and also converts a larger proportion of enzyme from fetal lung into a high molecular weight species. The enzyme from adult lung can be dissociated into a form in fetal lung. The dissociated species can be converted back to a high molecular weight form in the presence of phosphatidylglycerol.


(+/-)-[3H]Epinephrine and (-)[3H]dihydroalprenolol binding to beta1- and beta2-noradrenergic receptors in brain, heart, and lung membranes. (+/-)-[3H]Epinephrine binds to beta-receptors in calf cerebellar and rat lung membranes in the presence of 1.0 mM pyrocatechol and 1.0 microM phentolamine, with dissociation constants at 4 degrees C of 11 nM and 24 nM, respectively. (+/-)-[3H]Epinephrine associates to equilibrium within 20 min in both tissues, and over 50% of the binding is rapidly dissociable. Inhibition of binding by agonists and antagonists is highly stereoselective, and the structure-activity relationships of adrenergic agents in inhibiting (+/-)-[3H]epinephrine binding suggest an interaction with beta2 type noradrenergic receptors. (-)-Isoproterenol has an apparent Ki of 2 nM, (-)-epinephrine is 1.5 to 3 times weaker, and (-)-norepinephrine is 30 to 60 times weaker. Salbutamol and terbutaline, selective beta2-agonists, are potent inhibitors of binding, as are several nonspecific antagonists. Properties of the sites labeled by (+/-)-[3H]epinephrine in calf cerebellum and rat lung are closely similar. (-)-[3H]Dihydroalprenolol binding in calf cerebellum and rat lung also shows beta2 characteristics. Antagonists have similar potencies in inhibiting (-)-[3H]dihydroalprenolol and (+/-)-[3H]epinephrine binding in both tissues, but agonists are in general more potent inhibitors of (+/-)-[3H]epinephrine. Sodium and lithium selectively lower the affinity of (+/-)-[3H]epinephrine at its binding sites and the affinities of agonists, but not antagonists, at the (-)-[3H]dihydroalprenolol site. Specific (+/-)-[3H]epinephrine binding was not detectable in calf cortex and rat heart, where (-)-[3H]dihydroalprenolol binding suggests a beta1-receptor. A physiological significance of (+/-)-[3H]epinephrine binding is suggested by the strong correlation for agonists and antagonists between affinities in inhibiting binding, and in stimulating or inhibiting a beta-receptor-coupled adenylate cyclase in frog erythrocytes.


Altered metabolism of heparan sulfate in simian virus 40 transformed cloned mouse cells. Glycoconjugates have been analyzed from a family of closely related mouse cells: a parent clone and three daughter subclones, two of which expressed the simian virus 40 (SV40) T-antigen. The experimental procedure involved the simultaneous comparison by DEAE-cellulose chromatography of papain-digested macromolecules from the parent, labeled with [3H]glucosamine, and one of the daughter subclones, labeled with [14C]-glucosamine. Three cultures compartments (the medium, the cell surface trypsinate, and the cells) from the paired cell lines were combined at the earliest time during the harvesting of the cells. Heparan sulfate on the surface of cells and secreted into the medium from T-antigen-positive subclones was eluted at lower salt concentrations from the anion exchange column than that from the parent clone. In the viable trypsinized cells a marked reduction of heparan sulfate was detected in the T-antigen-positive subclones. These changes were highly reproducible, were observed during both logarithmic and stationary phase of growth, and neither change was observed in the T-antigen-negative sister subclone. The elution point of heparan sulfate from Sepharose 6B was unaltered. Ratios of 35S to 3H for heparan sulfate obtained from cells doubly labeled with [35S]sulfate and [3H]glucosamine were lower in the T-antigen-positive subclones. Similar changes for the 35S to 3H ratio of chondroitin sulfate were associated with only small alterations in elution from anion exchange columns. Kinetic experiments suggested a reduced rate of incorporation of [35S]sulfate with no change in turnover rate. A substantial portion of the labeled heparan sulfate was associated with the cell surface; in contrast most of the hyaluronic acid and a large proportion of the chondroitin sulfate was apparently secreted. Quantitative changes in hyaluronic acid labeling did not correlate with expression of T-antigen. Glycosaminoglycans left on the dish after detaching cells with ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid were nearly completely released by subsequent trypsinization. Cell detachment by trypsinization left an insignificant amount of labeled glycosaminoglycan on the dish surface. The alterations in heparan sulfate metabolism correlated with the expression of T-antigen and with the cells' ability to grow to high densities in monolayer culture, but not with growth in suspension in viscous medium. Tumorigenicity of the subclones was essentially the same as that of the parent clone.


Relationships between dephosphorylation and D to I conversion of rabbit skeletal muscle glycogen synthase. The relationship between dephosphorylation and D to I conversion of skeletal muscle glycogen synthase by synthase phosphatase was investigated using synthase preparations containing 1 to 3 mol of 32P/mol of subunit (90,000 g). Dephosphorylation was analyzed in terms of 32P release from the trypsin-sensitive and trypsin-insensitive phosphorylation regions of synthase. With synthase containing 1 to 2 mol of 32P/90,000 g, dephosphorylation of the trypsin-insensitive region correlated closely with D to I conversion and was more rapid than dephosphorylation of the trypsin-sensitive region. Synthase containing 3 mol of 32P/90,000 g was a relatively poor substrate for the phosphatase since dephosphorylation of both regions, as well as D to I conversion, was slow. With this species of synthase, glucose-6-P (0.1 mM) increased the rates of D to I conversion and dephosphorylation of trypsin-insensitive region. It is concluded that dephosphorylation of the trypsin-insensitive region is responsible for the conversion of synthase D to I.


Phosphorylation of NAD-dependent glutamate dehydrogenase from yeast. The NAD-dependent glutamate dehydrogenase from Candida utilis was isolated from 32P-labeled cells following enzyme inactivation promoted by glutamate starvation and found to exist in a phosphorylated form. Analysis of purified, fully active NAD-dependent glutamate dehydrogenase (a form) and inactive NAD-dependent glutamate dehydrogenase (b form) for alkalilabile phosphate revealed that the a form contained 0.09 +/- 0.06 mol of phosphate/mol of enzyme subunit and b form 1.25 +/- 0.06 mol of phosphate/mol of enzyme subunit. Phosphorylation caused a 10-fold reduction in enzyme specific activity. Dephosphorylation (release of 32P) and enzyme reactivation occurred on incubation with cell-free yeast extracts, indicating the presence of a phosphoprotein phosphatase in such preparations.


Protein is linked to the 5' end of poliovirus RNA by a phosphodiester linkage to tyrosine. Purification and partial characterization of the poliovirus RNA-linked protein (VPg) are described. VPg has been freed from the RNA by ribonuclease digestion and phenol extraction. Gel filtration chromatography of VPg-pUp (labeled with 32P) in 0.5% sodium dodecyl sulfate or 6 M guanidine HCl indicates that it has a molecular weight of about 12,000. VPg is bound to the 5' end of poliovirion RNA by a phosphodiester bond between a tyrosine residue in the VPg molecule and the 5'-terminal uridine. After acid hydrolysis of [3H]tyrosine-labeled VPg-pU, free tyrosine can be released by venom phosphodiesterase. Acid hydrolysis of VPg-p labeled with either 32P or [3H] tyrosine yields tyrosine-phosphate. There appears to be only 1 tyrosine residue per VPg molecule.


alpha-adrenergic receptor in rat heart. Effects of thyroidectomy. The effects of thyroid status on alpha-adrenergic receptors in the rat myocardium were investigated. The potent antagonist [3H]dihydroergokryptine was used to identify alpha-adrenergic receptors in rat heart particulate and sarcolemmal fractions. Administration of triiodothyronine to thyroidectomized rats decreased specific binding to alpha-adrenergic receptors in heart particulate and sarcolemmal fractions by 41% and 45%, respectively. Scatchard analysis revealed that the cardiac sarcolemmal fraction from thyroidectomized rats contained 29.3 fmol/mg of protein, as compared with 17.0 fmol/mg of protein found in the heart preparation of thyroidectomized rats treated with triiodothyronine. The equilibrium dissociation constants for the interaction of receptors with dihydroergokryptine were similar (about 1.5 nM) in the heart sarcolemmal fractions derived from these two groups of rats. The results of this study demonstrate that thyroid hormone can regulate the number of cardiac alpha-adrenergic receptors. In addition, there appears to be a reciprocal relationship between alpha-adrenergic and beta-adrenergic receptors in the rat myocardium.


Effect of tunicamycin on the secretion of serum proteins by primary cultures of rat and chick hepatocytes. Studies on transferrin, very low density lipoprotein, and serum albumin. Using rat or chick hepatocyte monolayers, we have studied the effect of tunicamycin, a specific inhibitor of protein glycosylation, on the synthesis and secretion of serum proteins. Tunicamycin inhibited glucosamine incorporation into rat liver transferrin and the apoprotein B chain of chick liver very low density lipoprotein (VLDL) by 75 to 90%. In contrasts, amino acid incorporation into these two glycoproteins, as well as into the normally unglycosylated proteins, rat serum albumin and apoprotein A of chick liver VLDL, was decreased by only 10 to 25% in the presence of the antibiotic. Despite the inhibitory effect of tunicamycin on glycosylation, secretion of all four proteins was virtually unimpaired. Thus, the carbohydrate moieties of rat liver transferrin or apoprotein B of chick liver VLDL do not appear to play an essential role in the secretion process.


Regulation of adenosine 3':5'-monophosphate efflux from animal cells. Cyclic AMP efflux was measured following hormonal stimulation of adenylate cyclase in a variety of animal cells including C-6 rat glioma cells, WI-38 human fibroblasts, and avian erythrocytes. Using a variety inhibitors of mitochondrial function and glycolysis, a correlation was noted between cellular ATP levels and the rate of cyclic AMP efflux in all cells examined. A relationship between the efflux rate and the magnitude of the membrane potential was not observed. Pharmacological agents which inhibited cyclic AMP egress in these cells without reducing ATP levels included several prostaglandins (A greater than B greater than E greater than F) and probenecid. The characteristics of the cyclic AMP efflux system resemble those of the organic anion transport system.


Chinese hamster lung cell polysomes direct the synthesis of a single molecular weight species of procollagen alpha chains. Polysomes prepared from cultured Chinese hamster lung cells direct the synthesis of procollagen alpha chains in an heterologous cell-free system containing the postribosomal supernatant fraction prepared from wheat germ. Total protein synthesis requires both subcellular components and an exogenous energy source, and is inhibited by the antibiotics puromycin and aurin tricarboxylic acid. The ratio of collagenase-digestible to nondigestible material produced depends upon the wheat germ and not the polysome level in the reaction. Under optimal conditions, a significant fraction of the total product migrates on denaturing sodium dodecyl sulfate-polyacrylamide gels as a single molecular weight collagenase-digestible species corresponding in size to the procollagen alpha chain (Mr approximately equal to 170,000). Approximately one-third of this high molecular weight material represents products whose synthesis results from cell-free mRNA initiation, and no distinct product larger than the 170,000-dalton material is observed. These studies confirm the initial observation that collagen represents one of the major gene products of Chinese hamster lung cells and demonstrate the usefulness of this cell line for the study of mammalian collagen biosynthesis.


Localization of the binding site for cell attachment in the alpha1(I) chain of collagen. Certain cells such as CHO (Chinese hamster ovary) and fibroblasts attach to a substrate of type I collagen via proteins that link the cell surface to collagen. Serum contains a glycoprotein, c-CAP (collagen cell attachment protein), that can mediate this adhesion. Previously, we established that alpha1(I)-CB7 (cyanogen bromide peptide 7), which lies within residues 552 to 822 of collagen, contains a major binding site for c-CAP(alpha1(I)-CB7 has been renumbered to account for an additional triplet recently identified at residue 613 (P. P. Fietzek and R. W. Glanville, manuscript in preparation). Now we have examined the ability of various peptides derived by proteolytic digestion of alpha1(I)-CB7 to bind to c-CAP based upon their ability to inhibit cell attachment to collagen. The binding site lies within residues 757 to 791. It is likely that this is the sole binding site in the alpha1(I) chain since cleavage of the bond between residues 775 and 776 in the alpha1(I) chain destroys cell attachment activity.


An H3 histone-specific kinase isolated from bovine thymus chromatin. A substantial portion of the histone phosphorylating activity of bovine thymus chromatin can be isolated by extraction in 0.2 M NaCl. The specificity of this extract for either free histones or washed chromatin substrates was compared. The salt-extracted kinase enzymes favor H2b as the major acceptor when whole free histone is the substrate and H3 when the substrate is intact chromatin. The H3 kinase activity of bovine thymus tissue has been purified free from other detectable histone kinase activities by ammonium sulfate fractionation and is highly specific for H3 histone when assayed either with chromatin or isolated whole histone. The activity is cAMP-independent. Tryptic peptide mapping of the labeled H3 histone reveals a single site of phosphorylation. This site appears to be identical with the major site of metaphase-associated H3 phosphorylation in hepatoma tissue culture cells. No corresponding H3 phosphorylation has been detected in thymus tissue in vivo.


Studies of cAMP metabolism in cultured hepatoma cells: presence of functional adenylate cyclase despite low cAMP content and lack of hormonal responsiveness. The ability of isoproterenol, glucagon, PGE1 and cholera toxin to stimulate the synthesis of cAMP and protein kinase activity in line of liver cells (BRL) and a line of rat hepatoma cells (H35) has been determined. The concentration of cAMP in BRL cells (approximately 10 pmoles/mg protein) is in the range reported for other cultured cell lines but H35 cells contain extraordinarily low amounts of this cyclic nucleotide (approximately 0.05 pmoles/mg protein). Isoproterenol and PGE1 caused an increase in cAMP content, and protein kinase activation in BRL cells, although glucagon was ineffective. H35 cells, in contrast, were completely insensitive to all hormonal agonists. Despite this fact, cholera toxin was able to produce a marked increase in cAMP content, adenylate cyclase activity and protein kinase activation in H35 cells. binding studies with [125 I]-iodohydroxybenzylpindolol, a specific beta-adrenergic receptor antagonist, revealed that each H35 cell possesses fewer than 10 beta-adrenergic receptors whereas BRL cells contain 2-5,000 receptors per cell. The low level of cAMP in H35 cells appears to result from a combination of totally unstimulated adenylate cyclase and apparently elevated phosphodiesterase activities.


Studies on the association of the Epstein-Barr virus genome and human chromosomes. We have used human-mouse somatic cell hybrid to study the association between the EBV genome and the cellular genome. Attempts were made to identify a specific human chromosome(s) with which the EBV genome is associated. Our data suggest that at least in the mouse fibroblast/Burkitt lymphoma hybrid combination studies, the EBV genome is not associated with any specific human chromosome.


Hormonal activation of adenylate cyclase in mouse melanoma metastatic variants. The ability of melanocyte stimulating hormone (MSH), adrenocorticotropic hormone (ACTH), and prostaglandin E1 (PGE1) to stimulate the accumulation of cyclic AMP was examined in intact mouse melanoma cells of varying metastatic potential. F1 cells (low metastatic potential) had significantly greater cyclic AMP levels in response to all three hormones than F5 (intermediate metastatic potential) and F10 (high metastatic potential) cells. The ranking of the response was as follows: MSH, F1 greater than F5 greater than F10, ACTH, F1 greater than F5 greater F10, PGE, F1 greater than F10 greater F5. In contrast to the above, the degree of hormonal stimulation of adenylate cyclase in broken cell preparations was virtually identical in all three melanoma cell lines. Control enzyme activity was depressed in both F5 and F10 relative to F1. The conflicting results between studies of intact vs. broken cell preparations could not be explained by increased cyclic AMP phosphodiesterase activity in F5 and F10 cells. We conclude that as the melanoma cells increase in metastatic potential, there is a significant loss in the ability of their cyclic AMP system to respond appropriately to hormonal stimuli.


The role of phosphodiesterase in aggregation of Dictyostelium discoideum. The role of cAMP phosphodiesterase in the cAMP-mediated aggregation of the cellular slime mould Dictyostelium discoideum was investigated with a morphogenetic mutant defective in phosphodiesterase production. Mutant cells become capable of aggregating normally when incubated in the presence of exogenous phosphodiesterase isolated from Idictyostelium or rat brain. Direct contact between enzyme and the cell membrane is not required for this phenotypic suppression. The aggregateless character of this strain presumably results from an over-accumulation of cAMP in the extracellular medium since aggregation can be induced in the absence of added phosphodiesterase under conditions facilitating diffusion of the nucleotide. This suggests that phosphodiesterase is not involved in the generation or recognition of cAMP signals, but that the enzyme is essential in the control of the cAMP signal-to-noise ratio.


Immune electron microscopy of avian infectious bronchitis virus serotypes. An immune electron microscopy agglutination technique in which emphasis is placed upon the importance of antigen-antibody equivalence has been developed as a possible method for the serotyping of avian infectious bronchitis viruses. The Connecticut and Massachusetts 41 serotypes were used as a model system. Stock virus concentrations were standardized by physical particle counts of virions sedimented directly onto electron microscope specimen grids. Suspensions containing approximately 150 virions per grid square were allowed to react with dilutions of homologous and heterologous antisera. Virions in these constant virus-variable serum mixtures were sedimented directly onto electron microscope specimen grids, and the relative degree of aggregation per grid was determined from the mean percent aggregation of five randomly selected grid squares. In homologous assays, regions of relative antibody excess, of equivalence, and of relative antigen excess were clearly evident. At equivalence, the mean percent aggregation was significantly higher than in the regions of relative antibody or antigen excess. In the heterologous systems, the degree of aggregation differed little from that of the virus controls containing no antiserum.


Hemoagglutination and hemagglutination inhibition tests with enterovirus type 70. Human type "O," guinea pig, and chicken erythrocytes were agglutinated by enterovirus type 70 at 4 degrees C or room temperature. A hemagglutination inhibition test, using human "O" erythrocytes, is described for the serological diagnosis of acute hemorrhagic conjunctivitis caused by enterovirus type 70.


Noncultivable viruses and neonatal diarrhea: fifteen-month survey in a newborn special care nursery. During a 15-month period of surveillance, diarrhea developed in 257 of 913 babies (28%) admitted within 2 hours of birth to a special care nursery in Melbourne, Australia. Diarrhea was seasonal, affecting a maximum of 43% of babies admitted during one winter month (July) and a minimum of 13% of babies admitted during one summer month (December). Diarrhea was no more frequent nor more severe in babies of very low birth weight or of very early gestational age. Two noncultivable viruses were located by electron microscopy in feces from babies with or without diarrhea. Excretion of a reovirus-like particle (rotavirus, duovirus, human reovirus-like agent, infantile gastroenteritis virus) was temporally related to diarrheal symptoms. Asymptomatic infection with this virus also occurred. A 28-nm virus-like particle was excreted by some babies, but it could not be implicated on epidemiological grounds in the etiology of the diarrhea. Rotavirus infection may be an important cause of endemic diarrhea in nurseries for the newborn. Infection may be difficult to control or eradicate, since it is often asymptomatic and may be influenced by infection in the community at large.


Selective uptake of the synthetic amino terminal fragment of bovine parathyroid hormone by isolated perfused bone. Studies from our laboratory have shown that the metabolic clearance rate of carboxy terminal immunoreactive parathyroid hormone (i-PTH) can be accounted for by extraction of i-PTH by liver and kidney. In contrast, there was no demonstrable hepatic uptake of the synthetic amino terminal bovine PTH fragment (syn b-PTH 1-34) and the kidney accounted for only 45% of the metabolic clearance rate of amino terminal i-PTH. This suggested that another major site, presumably bone, played a role in the metabolism of syn b-PTH 1-34. Extraction of i-PTH by isolated perfused bone was studied during infusion of purified bovine PTH (b-PTH) 1-84 or syn b-PTH 1-34. In five studies during infusion of syn b-PTH 1-34 the arteriovenous difference for i-PTH across bone was 36%. In contrast, no significant uptake of carboxy terminal i-PTH was observed in nine studies during infusion of b-PTH 1-84. In addition, when H(2)O(2)-oxidized (biologically inactive) syn b-PTH 1-34 was used no arteriovenous difference was observed. These findings correlated with the ability of these PTH preparations to stimulate cyclic AMP production by the perfused bone. Syn b-PTH 1-34 increased cyclic AMP production at perfusate PTH concentrations of 1-5 ng/ml, whereas b-PTH 1-84 evoked only a minimal response at concentrations of 10-20 ng/ml. We conclude that bone is a major site of metabolism of the amino terminal PTH fragment, syn b-PTH 1-34. In addition, these data suggest that cleavage of the intact hormone, with the production of amino terminal PTH fragments by peripheral organs (liver and kidney), may play a major role in the regulation of PTH effects on bone.


Activation of adenylate cyclase by heat-labile Escherichia coli enterotoxin. Evidence for ADP-ribosyltransferase activity similar to that of choleragen. Highly purified, polymyxin-released, low molecular weight Escherichia coli heat-labile enterotoxin (LT) catalyzed the hydrolysis of NAD to ADP-ribose and nicotinamide. This NAD glycohydrolase activity was stimulated by dithiothreitol and was independent of cellular components. Nicotinamide formation was enhanced by arginine methyl ester > d-arginine congruent with l-arginine congruent with guanidine. A 20-fold increase in activity was noted with arginine methyl ester, and maximal activity again required dithiothreitol. When the reaction was initiated with toxin, a delay was observed before a constant rate was established. The reaction products found after incubation of [adenine-U-(14)C]NAD and l-[(3)H]arginine or unlabeled arginine methyl ester with the enterotoxin had mobilities on thin-layer chromatograms similar to the reaction products obtained after incubation of choleragen with these substrates and are consistent with the formation of ADP-ribose-l-arginine and ADP-ribose-l-arginine methyl ester, respectively. Both toxins, which catalyze the NAD-dependent activation of adenylate cyclase, thus appear to possess NAD glycohydrolase and ADP-ribosyltransferase activities. Although the activities of both toxins are dependent on dithiothreitol, Escherichia coli enterotoxin exhibited optimal activity in Tris (Cl(-)) (pH 7.5) and was inhibited by high concentrations of potassium phosphate (pH 7.0) or low pH (sodium acetate, pH 6.2). It appears that the optimal assay conditions as well as the kinetic constants for the reactants differ from those previously noted with choleragen. It is probable therefore that although the two toxins catalyze similar reactions, they differ in primary structure. The presence of transferase and glycohydrolase activities in structurally distinct toxins that activate adenylate cyclase strengthens our hypothesis that the ADP-ribosylation of arginine is a model for the NAD-dependent activation of adenylate cyclase; activation may result from ADP-ribosylation of the cyclase itself or of a protein that regulates its activity.


Bone marrow-derived lymphoid cell lines from patients with agammaglobulinemia. In vitro infection with Epstein-Barr virus of bone marrow-derived (B) lymphocytes from blood of patients with X-linked or common varied agammaglobulinemia resulted in the establishment of long-term B-lymphoid cell lines (LCL). LCL were established only with B lymphocytes from patients whose B cells failed to respond to in vitro mitogenic stimulation. In contrast, lymphocytes from patients without small B lymphocytes or with B cells that synthesized but failed to secrete Ig failed uniformly to establish LCL. Analysis of the "nonsecretory" B lymphocytes demonstrated the presence of receptors for C3b and C3d as well as surface Ig, but absence of detectable receptors for Epstein-Barr virus. All of the six cell lines that were established formed rosettes with EAC3 but not with sheep erythrocytes. Four of the six LCL were principally surface IgD bearing and did not synthesize detectable Ig for secretion. Two of the cell lines were indistinguishable from cell lines from normal individuals: they had surface IgG, A, M, and D and synthesized and secreted Ig.


Interaction of diphenylhydantoin (phenytoin) and phenobarbital with hormonal mediation of fetal rat bone resorption in vitro. Chronic administration of high doses of anticonvulsant drugs frequently produces classic osteomalacia with bone histologic changes characteristic of increased parathyroid hormone (PTH) effect in man. However, several reports have documented defects in calcified tissue metabolism suggestive of an end-organ resistance to PTH after chronic anticonvulsant drug therapy. To examine the direct action of anticonvulsant drugs on bone resorption, we investigated the effects of diphenylhydantoin (phenytoin) (DPH) (100-200 mug/ml) and phenobarbital (10-400 mug/ml) on basal and hormonally mediated resorption 5-day cultures of fetal rat forelimb rudiments. In this system both drugs significantly inhibited basal and PTH-stimulated (45)Ca and [(3)H]hydroxyproline release, as well as 1,25-dihydroxyvitamin D(3)-stimulated (45)Ca release. The effects of DPH and phenobarbital were additive, with DPH exhibiting a several-fold more potent inhibitory effect than phenobarbital. Whereas DPH exhibited a striking synergism with the inhibitory effects of human calcitonin (HCT) on PTH-induced resorption, the effect of phenobarbital was merely additive to that of HCT. PTH and PTH plus HCT-induced increases in bone cyclic AMP (cAMP) content were significantly inhibited by DPH but not by phenobarbital. However, in contrast to effects on (45)Ca release, DPH inhibition of cAMP generation was not accentuated in the presence of HCT. It is concluded that: (a) both DPH and phenobarbital can directly inhibit basal and hormonally stimulated bone resorption, with DPH being much more potent in this regard; (b) DPH appears to inhibit bone resorption via a cAMP-independent mechanism and has an additional suppressive effect on PTH-induced cAMP generation; and (c) the synergistic interaction of DPH and HCT in inhibiting (45)Ca release occurs at a site independent of cAMP generation.


Ectopic ACTH syndrome: clinicopathological correlations. Ten out of 164 cases of bronchogenic carcinoma showed pathological evidence at necropsy of the ectopic ACTH syndrome. All occurred in association with oat-cell carcinoma, constituting 19% of that group. The pathological features consisted of adrenocortical hyperplasia confined to the zona fasciculata and Crooke's hyaline change in the pituitary. Immunoperoxidase stainable ACTH was detected in the pituitary but not in the carcinoma tissue, a surprising finding, which may be due to the different nature of ACTH present in tumour tissue. The ectopic ACTH syndrome was diagnosed ante mortem in only four out of 10 patients on the basis of hypokalaemia and metabolic alkalosis. The lack of clinical pointers in all but terminal cases is discussed as well as possible measures for earlier diagnosis.


A case of type III hyperlipoproteinaemia studied by acrylamide gel gradient electrophoresis. A case of type III hyperlipoproteinaemia has been investigated before and after treatment, and the unusual serum lipoprotein patterns obtained by molecular exclusion or pore limit electrophoresis on acrylamide gradients have been compared.


Demonstration of saturation kinetics in the intestinal absorption of vitamin C in man and the guinea pig. Intestinal absorption of ascorbic acid is believed to be mediated through a sodium-dependent active transport process in man and in the guinea pig, both species having a nutritional requirement for the vitamin. Vitamin C transport was studied in man and in the guinea pig by in vivo intestinal perfusion of concentrations of vitamin C ranging from physiologic to clearly pharmacologic levels. Triple lumen intestinal perfusion of seven human volunteers with vitamin C concentrations ranging from 0.85 to 11.36 mM demonstrated saturation kinetics of absorption with a Km = 5.44 mM. Net secretion of water was observed in three of seven humans with the highest (11.36 mM) concentration of vitamin C. Perfusion of isolated segments of guinea pig intestines with intact blood supply also revealed saturation kinetics (Km = 5.54 mM) in the range of 1.42 to 56.8 mM vitamin C but linear absorption below this range. The phenomenon of decreased water absorption noted with incremental vitamin C dose in human volunteers could not be reproduced in the guinea pig, nor were the intestinal tissue levels of cyclic AMP and GMP increased by high-dose vitamin C in this species. This study suggests that "megavitamin" doses of vitamin C (greater than 1 Gm) are probably not as efficiently absorbed as smaller multiple doses of the vitamin. Intestinal secretion of water may contribute to the diarrhea which is the most common side effect of large doses of vitamin C. The guinea pig is a useful but limited model for vitamin C absorption in man.


Surgical treatment of submucosal tumors of the hard palate. Two cases have been presented to illustrated to illustrate the principles of total excisional biopsy for palatal tumors and the postoperative management of the resulting defect with the use of palatal splints. Total excisional biopsy is curative for benign tumors and for some malignant-grade tumors, and is the preferred method of treatment when possible. Total excision of the lesion with adequate margins negates the possibility of seeding the tumor into surrounding tissue or of leaving behind residual tumor.


A review of marijuana in relation to stress-response mechanisms in the dental patient. A comprehensive review of the established physiologic and metabolic effects of marijuana in relation to cardiovascular physiology and stress-response mechanisms has been presented. This information is related to the preoperative and postoperative treatment of the dental patient who smokes marijuana habitually. Marijuana is not a benign drug. Its vasoactive, cardiotropic, steroidogenic, and enhanced sympathoadrenal stress-response effects justify caution when administering atropine or epinephrine-containing products to the habitual user of marijuana. More clinical as well as laboratory research is needed to elucidate the speculation regarding a drug so frequently misused.


Cyclic 3',5'-nucleotide phosphodiesterase; cytochemical localization in rat renomedullary interstitial cells. The localization of cyclic 3', 5' -nucleotide phosphodiesterase activity in rat renal papillae was examined by utilizing cytochemical methods. Renal medullary interstitial cells had predictable phosphodiesterase activity predominantly on the cytoplasmic border of dilated cisternal membranes. Cells of the collecting tubule and loop of Henle contained diffuse reaction product. Capillaries had reaction product localized in pinocytic vesicles. Addition of theophylline resulted in no deposition of reaction product in interstitial cells and in cells of the collecting tubule and loop of Henle, suggesting an inhibition of phosphodiesterase activity. Since the membranes of dilated cisternae of renal medullary interstitial cells have been shown to be related to prostaglandin synthesis and probably to the anti-hypertensive function of these cells, the finding of phosphodiesterase activity on these membranes suggests a possible role of cyclic AMP in these two functions.


Glucocorticoid receptors and glucocorticoid-sensitive secretion of neutral proteinases in a macrophage line. A continuous line of mouse macrophages (P388D1) has been shown to secrete elastase, collagenase, and plasminogen activator at activities comparable to those of macrophages elicited by an inflammatory stimulus in vivo. At physiologic concentrations anti-inflammatory glucocorticoids selectively and reversibly inhibited secretion of the three proteinases but did not inhibit secretion of lysozyme, a constitutive enzyme produced by the P388D1 cells. The secretion of the neutral proteinases was inhibited 50% by 2 to 10 nM dexamethasone. Proliferation of the macrophages was also glucocorticoid sensitive. The P388D1 macrophages contained about 4000 saturable glucocorticoid-binding sites per cell. Concentrations of hormone saturating the high affinity receptor site (for dexamethasone the dissociation constant for steroid-receptor binding, Kd, was 4 nM) correlated well with concentrations inhibiting secretion of the proteinases. Only glucocorticoids and progesterone competed for binding to the specific receptors. Temperature-sensitive translocation of hormone-receptor complexes from "cytoplasm" to nucleus similar to that found with rat thymocytes was demonstrated. Thus, the interaction between glucocorticoids and the P388D1 cell line provides a model for the regulation of macrophage secretion of neutral proteinases under normal and stress conditions.


Independent stimulation of motility and the oxidative metabolic burst of human polymorphonuclear leukocytes. It has recently been suggested that a single stimulus to the membrane of the polymorphonuclear neutrophil leukocyte (PMN) produces a sequential, stereotyped response involving motility, degranulation, and the oxidative metabolic burst, and conversely, that the chemotactic response is dependent upon the stimulation of the hexose monophosphate shunt (HMPS). We have used small, synthetic substances, known to cause either increased motility or the metabolic burst, to examine whether these events can be stimulated independently. Phorbol myristate acetate (PMA) is a surface active agent that causes marked stimulation of iodination, superoxide production, chemiluminescence, and the HMPS. Such stimulation by PMA did not alter random or directional motility of PMN in the chemotaxis-under-agarose assay. Also, preincubation of PMN with PMA did not deplete their energy source for chemotaxis as demonstrated by a normal chemotactic response to zymosan activated serum. N-formylmethionyl peptides (f-met-phe, f-met-leu-phe) caused a dose-related stimulation of random and directional motility of PMN, but only a very slight stimulation of the HMPS, protein iodination, superoxide production, or chemiluminescence, and this minimal response occurred at more than 1000 times the concentration needed for stimulation of motility. These results indicate that stimulation of motility in the metabolic burst may involve separate events at the membrane of the PMN and that the events are not necessarily interdependent.


Altered subcellular and submitochondrial localization of CTP:phosphatidate cytidylyltransferase in the Morris 7777 hepatoma. The subcellular and submitochondrial localization of CTP:phosphatidate cytidylyltransferase is altered in the Morris 7777 hepatoma. Mitochondria in this poorly differentiated tumor are the principal sites of CDP-diacylglycerol synthesis, in contrast to normal rat liver where the endoplasmic reticulum is most active. This enzyme activity was increased 17-fold in the outer mitochondrial membrane, and a 22% increase was noted in the inner mitochondrial membrane of the 7777 hepatoma as compared with the corresponding fractions from normal rat liver. Increased mitochondrial CTP:phosphatidate cytidylyltransferase was present in six other Morris hepatomas, but it was not found in fetal rat liver mitochondria, suggesting that rapid growth alone is not responsible for the difference. Evidence is presented which indicates that mitochondrial lipid degradation is similar in normal liver and the 7777 hepatoma, in vitro. The increased activity of CTP: phosphatidate cytidylytransferase is thought to be responsible in part for the moderately increased diphosphatidylglycerol content of 7777 hepatoma mitochondria.


Protein kinase-mediated phosphorylation of a purified sterol ester hydrolase from bovine adrenal cortex. Sterol ester hydrolase (cholesterol esterase, E.C. 3.1.1.13) of bovine adrenal cortex has been extensively purified by ammonium sulfate fractionation, acid precipitation, hydroxylapatite chromatography, and Sephadex G-200 chromatography. During the purification sequence, the hydrolase activity was purified free of endogenous protein kinase. With this purified preparation, activation by cyclic AMP and ATP-Mg2+ did not occur unless exogenous protein kinase was included in the activating system. Using [gamma-32P]ATP, the transfer of the terminal phosphate to the enzyme protein was demonstrated by three separate experimental approaches. With pooled fractions from Sephadex G-200 chromatography, significant binding of 32P by the enzyme protein was observed only in the presence of exogenous protein kinase. Time course studies disclosed a close concurrence between the extent of activation of the purified enzyme by cyclic AMP-dependent protein kinase and the level of 32P transfer from [gamma-32P]ATP to the enzyme protein. Finally, assays carried out during Sephadex G-200 chromatography showed a correspondence in the peaks for activated sterol ester hydrolase and for 32P binding by protein. The data confirm that activation of adrenal sterol ester hydrolase by cyclic AMP and ATP-Mg2+ involves protein kinase-catalyzed phosphorylation of the enzyme protein.


ACTH-induced hydrolysis of cholesteryl esters in rat adrenal cells. Rat adrenal cortical cells have been prepared by collagenase dissociation of trypsin-treated adrenal tissue. The content and compositions of cholesteryl ester, phospholipid, and triglyceride fatty acids compare favorably with those of undissociated rat adrenal tissue. During 2-hour control incubations of adrenal cortical cells, steroidogenesis was not detected, and the levels of sterol ester, phospholipid, and triglyceride fatty acids were not significantly altered. Incubations with adrenocorticotropic hormone (ACTH) resulted in coricosterone production and significant depletions of sterol ester and triglyceride fatty acids, but not of phospholipid fatty acids. Although all fatty acid esters of cholesterol were hydrolyzed under these conditions, the greatest contributions to the net decrease in sterol esters were by oleate, arachidonate, and adrenate. Incubations with dibutyryl cyclic adenosine monophosphate (0.5 mM) resulted in significantly greater levels of corticosterone production than did ACTH (250 muunits), but the effects on cellular lipids were comparable to those seen with the tropic hormone. This study represents the first demonstration of hormone-induced hydrolysis of sterol esters in an in vitro cell suspension system. The results are discussed with respect to hormone-sensitive sterol ester hydrolase of adrenal cortex, and to the role of endogenous cholesteryl esters in the steroidogenic pathway.


Direct measurement of apoprotein B specific activity in 125I-labeled lipoproteins. A method for determining apoprotein B specific activity in radioiodinated lipoproteins is described and validated. It utilizes organic solvents and tetramethylurea in the isolation of apoprotein B from other radiolabeled contaminants, both lipid and protein, in exogenously labeled VLDL. The contaminants are also removed from those lipoprotein classes subsequently derived from VLDL, namely IDL and LDL. The procedure requires approximately 50 microgram of apoB per analysis, allowing specific activity determinations in triplicate on 3-ml plasma samples with a standard error of less than 6%. Finally, data from a study of apoprotein B turnover in VLDL, IDL, and LDL in a human subject is presented to demonstrate the potential of this method in further elucidating the kinetic interrelationships between these lipoprotein classes.