Cervical cancer control: a study of morbidity and mortality trends over a twenty-one-year period. Data from a 21-year period are presented to evaluate the effects of a mass cytologic screening program on uterine cancer morbidity and mortality in Louisville, Jefferson County, Kentucky. The success of screening was greatest in the younger age groups. There was a fall-off after age 45 years, especially in those age 60 years or older. Women at highest risk for cervical cancer, in the low socioeconomic quartile, had a better initial screening rate than the two middle-income quartiles, and had the highest rate of all women for subsequent rescreening. The greatest decrease in both morbidity and mortality was in women under the age of 50 years. Women age 30-39 and 50-59 years benefitted the most, as measured by mortality, with a decrease of 70.8 and 69.0%, respectively. There was no change in mortality rates for those age 70 years or older. Although the average annual age-adjusted rate increased from 13.2 to 15.3/100,000 women over the 21-year period.


Population cancer screening.

Population cancer screening. Controlled trials to evaluate mass screening programs for cancer detection have been singularly lacking. High cost, lack of medical manpower, and low yield have contributed to this problem. A new program in cancer detection (CANSCREEN) has been developed jointly by The Fox Chase Cancer Center in Philadelphia and the Preventive Medicine Institute in New York City. This program attempts to provide a quality cancer-detection examination with increased cost effectiveness. Features include: 1) a self-administered questionnaire on medical history, symptoms, and risk factors; 2) nonphysican examiners; 3) risk-facotr analysis with a predetermined decision logic to determine type and periodicity of examination; and 4) primary intervention (health education). This collaborative program between two institutions in two cities demonstrates the feasibility of introducing similar programs elsewhere. A data base shared by cooperating centers permits information on all patients to be used for evaluation of new techniques, end results, etc. A randomized controlled trial has been designed to evaluate the effectiveness and efficiency of the questionnaire alone and of the questionnaire and examination.


Parathyroid adenoma and nonmedullary thyroid carcinoma. The incidence of thyroid carcinoma in patients with hyperparathyroidism has been reported as higher than expected. We record our experience with 272 cases of primary hyperparathyroidism. Malignant thyroid lesions were found in 11.4%. We conclude that this is more than a fortuitous association.


Melanomas of the lower extremity among native Puerto Ricans. The clinical features of 57 melanomas of the lower extremity occurring in native Puerto Ricans were studied. Whereas most melanomas of the lower extremity among Caucasians occur above the ankle, the group studied showed a predilection for the foot, particularly in the minimally pigmented zones (sole, heel, and nail bed), a distribution similar to that reported in black patients. The possibility that pigment-deficient areas may represent the target organ for sunligh-induced melanomas in Negroes and dark-skinned subjects is discussed.


Second-look operation in ovarian carcinoma: postchemotherapy. One hundred and three patients with advanced ovarian cancer underwent a second-look operation following chemotherapy. Patients should have ten or more courses of chemotherapy before second-look operation. Those patients with no evidence of disease at second laparotomy should discontinue their chemotherapy. Patients who continued chemotherapy after the second-look operation did better than those who were treated with radiation after surgery. Only patients with clinical remission benefit from the second-look procedure. Advantages of second-look operations following chemotherapy are discussed.


The investigation and management of Hodgkin's disease in the pregnant patient. The management of Hodgkin's disease during pregnancy is reviewed in the light of experience from 15 patients. The modification of the investigative procedures during pregnancy is discussed and the use of the single-film (at 24 hours) lymphogram is described. The importance of an individual treatment policy for each patient, after careful consideration of the facts of the case, is emphasized and the dangers of inadequate therapy are stressed.


Chromosomal characteristics of malignant lymphoma in northern pike (Esox lucius) from the United States. Chromosome analyses were performed on tumor cells and normal hematopoietic cells of northern pike bearing malignant lymphomas. The normal pike karyotype contains 50 acrocentric chromosomes, among which the sex chromosomes are not identifiable by available techniques. Metaphases from 19 primary lymphomas showed a consistent pattern of chromosome anomalies consisting of 1 submetacentric marker, 1 minute marker, and 3 to 5 pairs of smaller-than-normal chromosomes, set within a mode of 50 and a range of 46 to 54. Occasional cells had 2 identical submetacentric markers. Cells from presumptively normal renal hematopoietic tissue in 41 lymphoma-bearing pike showed the euploid karyotype, with the exception of 2 cases in which some cells with the neoplastic pattern were found. This probably indicated occult neoplastic involvement of renal hematopoietic tissue, in spite of the absence of histological evidence of involvement. In many cases, some of the cells taken directly from lymphomas bore the normal karyotype. It is not known whether these cells were reactive lymphoid cells or lymphoma cells that had not developed those anomalies typically associated with the tumors. The findings do not permit a conclusion regarding agent versus cellular transmission. However, the pike lymphoma is an excellent example of a neoplasm with a karyotypic "signature."


Bacterial proteinaceous products (bacteriocins) as cytotoxic agents of neoplasia. Several bacteriocins, bacterial proteinaceous antibiotics, are shown to markedly inhibit the division of various established (neoplastic) mammalian cell lines. The bacteriocins tested originated from Escherichia coli, Pseudomonas aeruginosa, Vibrio cholerae, and Vibrio eltor. Using exponentially growing L60T mouse fibroblasts, the inhibitory effect was concentration dependent, and a growth inhibitory unit, equivalent to cytotoxic index 50, was established. Expression of toxicity as a function of duration of exposure to pyocin required 3 to 4 hr. DNA synthesis was inhibited and reflected the effects on growth inhibition. Maximal sensitivity to the bacteriocin was observed prior to mitosis in the G2 phase of the cell cycle.


Effect of defibrination on tumor growth and response to chemotherapy. Other investigators have demonstrated fibrin deposition in tumors. Experiments were therefore designed to test whether systemic defibrination would alter tumor growth or tumor response to chemotherapy with cyclophosphamide. Defibrination with Ancrod, a venom extract of Agkistrodon rhodostoma, did not significantly affect tumor sensitivity to chemotherapy. Similarly, defibrination plus fibrinolytic therapy with streptokinase did not affect responsiveness to cyclophosphamide. Long-term defibrination did not affect tumor growth. These results suggest three possible interpretations: (a) the coagulation system may not be important in tumor growth and response to chemotherapy; (b) adequate clearing of fibrin from the tumor was not accomplished in our experiments; or (c) other factors such as platelet deposition may be involved and platelet function was not inhibited by the therapies used in our experiments.


Conversion of the oncogenic 1-methylguanine 3-oxide to 3-hydroxy-1-methylxanthine. Deamination of the oncogenic 1-methylguanine 3-oxide occurs to a significant extent in rats to yield 3-hydroxy-1-methylxanthine and its metabolites. When 3-hydroxy-1-methylxanthine is administered, 1-methyl-8-methylthioxanthine can be recovered from urine and released from hepatic protein. No 1-methyl-8-methylthioguanine was detected in urine or bound to protein. There is no evidence of significant activation of 1-methylguanine 3-oxide by sulfotransferase, but deamination to the oncogenic 3-hydroxy-1-methylxanthine suffices to explain its oncogenicity.


Tumoricidal properties of mouse macrophages activated with mediators from rat lymphocytes stimulated with concanavalin A. Macrophage-activating factor (MAF) was obtained from cultures of normal F344 rat lymphocytes incubated with insoluble concanavalin A. The MAF rendered macrophages from normal C57BL/6 mice cytotoxic against the syngeneic B16 melanoma and the allogeneic AC 15091. At the same time, normal syngeneic or allogeneic embryo cells were unharmed, even in the presence of susceptible tumor cells. Optimal MAF levels followed incubation of lymphocytes for 48 hr with Sepharose-bound concanavalin A. A 2-hr incubation of macrophages with MAF was sufficient to initiate activation, providing that 46 hr were allowed to elapse before tumor cells were added. The MAF activity was enhanced after heating the supernatant to 199 degrees. Control experiments largely excluded the possibility that residual unbound concanavalin A caused the observed macrophage-mediated tumoricidal effects.


Lack of carcinogenicity of 4-, 5-, 6-, 7-, 8-, 9-, and 10-hydroxybenzo(a)pyrene on mouse skin. Seven phenols of benzo(a)pyrene (4-, 5-, 6-, 7-, 8-, 9-, and 10-hydroxybenzo(a)pyrene) were tested for carcinogenicity on mouse skin by application of 0.4 mumole of compound once every two weeks for 56 weeks. None of the seven phenols tested was carcinogenic to mouse skin, while treatment with the same dose of benzo(a)pyrene produced tumors in 92% of the treated animals. The lack of carcinogenicity of 7- and 8-hydroxybenzo(a)pyrene indicates that the strong carcinogenic activity previously reported for benzo(a)pyrene 7,8-oxide was not due to either phenolic isomerization product of this arene oxide.


Multiple sites of action of N-hydroxy-2-acetylaminofluorene rat hepatic nuclear transcription. This study attempts to identify the site(s) of action of N-hydroxy-2-acetylaminofluorene (N-OH-AAF) in relation to its inhibition of rat hepatic nuclear RNA synthesis. Two hr after N-OH-AAF injection (3 mg/100 g body weight), rat hepatic nuclear synthesis and nucleolar RNA synthesis in vitro were inhibited by 60 and 80%, respectively. When total nuclear RNA polymerases were solubilized and assayed in the presence of alpha-amanitin (3.2 mug/ml), only alpha-amanitin-sensitive activity was reduced (50%) by N-OH-AAF. Diethylamino-ethyl-Sephadex column chromatography confirmed this finding and further demonstrated that RNA polymerase II was the activity selectively inhibited. Since N-OH-AAF dramatically inhibited nucleolar RNA synthesis but had little effect on RNA polymerase I activity, per se, we therefore concluded that, in addition to its direct inhibitory effect on the enzymic function of RNA polymerase II, N-OH-AAF must also cause impairment of the nucleolar DNA template function.


Inhibition of DNA synthesis in normal and malignant human cells by triazinate (Baker's antifol) and methotrexate. Triazinate (TZT), a triazine folate antagonist, is a potent inhibitor of dihydrofolate reductase from mammalian cells. Because antitumor activity of triazinate in experimental tumors correlated closely with the in vitro inhibition of DNA synthesis in tumor cells derived from these tumors, we studied cells from patients with leukemia, solid tumor effusions, and cells from normal marrow to determine their in vitro sensitivity to TZT. DNA synthesis in cells from patients with acute leukemia was less sensitive to TZT than it was to methotrexate (MTX) at 2 X 10(-6) M concentration of the inhibitor, whereas the sensitivity was similar at 10(-5) M. This could be accounted for by the known greater sensitivity of dihydrofolate reductase to MTX than to TZT, and the observation that, whereas intracellular drug levels were similar at low (2 X 10(-6) M) extracellular concentrations of TZT or MTX, at the higher (10(-5) M) extracellular drug concentration intracellular TZT was greater than 3 times intracellular MTX. In vitro inhibition of DNA synthesis in cells obtained after patients were treated with TZT was correlated with drug serum concentration and with leukemia cell kill. The sensitivity of cells from solid tumor effusions to TZT was similar to the sensitivity to MTX. Since patients can tolerate doses of TZT five times higher than MTX with less toxicity, there may be advantage to the clinical use of TZT in some tumor cell types.


Abnormalities of complement and its components in patients with acute leukemia, Hodgkin's disease, and sarcoma. Whole complement and component titers were measured in patients with acute leukemia, Hodgkin's disease, and sarcoma. Serum samples were obtained from 42 consecutive patients and 11 healthy control subjects. Sera were frozen and maintained at -70 degrees until analyzed by hemolytic assay. Titers were normalized using a titer obtained from a single source of pooled human serum analyzed simultaneously with each patient sample to correct for day-to-day variation inherent in the assay technique. Significant elevations (p less than or equal to 0.05) of whole complement and C5, C8, and C9 were observed for each patient category, compared to controls. Forty-one of 42 patients had C9 titers greater than or equal to 2 S.D. above the mean titer for controls. Mean C3 and C7 titers were not elevated or depressed in any group. No clinical factors that correlated with abnormal complement or component titers were identified.


Multiphasic incidence of foreign body-induced sarcomas. Single or multiple plastic films (unplasticized vinyl chloride vinyl acetate copolymer) of different sizes and shapes were implanted s.c. in female CBA/H and CBA/H-T6 mice. Tumor incidence increased and accelerated with increased total surface area of multiple implants or with increased size of single implants. Tumor distribution curves over time were generally multiphasic. The profiles changed in proportionate relation to implant size. These findings indicate class differences between tumors according to latency. Since latency is known to be a predetermining characteristic of foreign body-induced tumors, class differences seem to exist at the originator cell level, reflecting diversity of intrinsic carcinogenic factors.


Distribution and excretion of (3H)vincristine in the rat and the dog. The distribution and excretion of tritiated vincristine were studies in the rat and the dog. Biphasic curves for the disappearance of the drug from blood were found in both species, with an initial half-life of approximately 15 min and a secondary half-life of approximately 75 min. Tissue levels were high in 1 hr in the rat and declined rapidly, except in the brain where very low levels of drug were found at all times. The bile was found to be the major route of excretion. The peak rate of excretion in bile was found to occur earlier in the rat (10 min) than in the dog (60 min). Rats given a higher dose of vincristine (1.0 mg/kg) excreted a larger percentage of the dose in the bile than rats given a lower dose (0.1 mg/kg). In rats given a low dose of vincristine (0.1 mg/kg), more than 85% of the drug was excreted in the feces and the urine over 72 hr. Less than 10% of the total radioactivity in the bile and urine was metabolites whereas, in the plasma, metabolites accounted for 40% of the total radioactivity.


The specificity of concomitant tumor immunity at large tumor volumes. Two antigenically distinct fibrosarcomas, designated BP-8 and BP-9, induced in syngeneic C3H/HeN mice by 3,4-benzo(a)pyrene were used to study tumor-specific immunity, concomitant tumor immunity and the effect of large tumor volumes on the loss of immunological reactivity. Two groups of mice were immunized to the BP-8 tumor by amputation of growing BP-8 isografts. One group was rechallenged with the BP-8 cells, and tumor growth was not noted. Both groups of mice then received an inoculum of BP-9 cells that grew to palpable tumors to the same extent as in control mice. BP-8-immunized mice bearing progressively larger PB-9 tumors were sacrified at varying intervals after the BP-9 isograft. Tumor weight was recorded as a percentage of total body weight and viable spleen cells from these animals were tested in vitro for cytotoxicity against BP-8 and BP-9 cells in the [125I]iododeoxyuridine microcytotoxicity assay. Spleen cells from untreated mice were used as controls. The mice with growing BP-9 tumors developed an immune reaction against the tumor antigens which increased with time from initial tumor isograft and with increasing tumor size up to a definite but variable limit. Cytotoxicity to BP-9 cells rose from 18% when the BP-9 tumor was not palpable to a maximum of 77% when the tumor represented 5 to 10% of the total body weight. Cytotoxicity to BP-9 fell progressively as tumor size exceeded 15% of the total body weight and approached the 10% background cytotoxicity of control lymphocytes to BP-9 cells, when the tumor weight achieved 25% of the animal's weight. Conversely, cytotoxicity of lymphocytes against the BP-8 tumor did not vary significantly and remained about 41 to 44% over the same interval even while specific reactivity to BP-9 cells significantly decreased. In addition, with time, lymphocyte-mediated cytotoxicity to the BP-8 tumor increased from 41 to 70% if the BP-8-immunized mice had been rechallenged with antigenically identical BP-8 cells prior to the BP-9 isograft. These data suggest that loss of immunoreactivity at large tumor volumes is tumor and, presumably, antigen specific. No evidence of a generalized immune paralysis was demonstrated, since the mice always maintained immunity to the BP-8 tumor despite progressive and lethal growth of the antigenically distinct BP-9 tumor.


Humoral regulation of cell-mediated immunity to syngeneic tumor. Inoculation i.p. of C57BL/6 mice with FBL-3 cells, a syngeneic Friend virus-induced leukemia, results in progressive growth of ascitic tumors; in contrast, s.c. inoculation of FBL-3 cells produces transient, localized tumor growth; the recipients are then subsequently resistant to further i.p. challenge of this tumor. Experiments were performed to study the effects of humoral factors that might be present in the ascitic fluid and that could affect the growth of the tumors and the host immune response. It was found that ascitic fluids obtained from various murine tumors could indeed promote the s.c. growth of FBL-3 cells. Furthermore, administration of these ascitic fluids was found to suppress the induction of both the primary and secondary cell-mediated cytotoxic responses to FBL-3 cells in vivo and in vitro and to inhibit the effector phase of these cell-mediated cytotoxic reactions in vitro. These studies indicate that the ascitic fluids obtained from tumor-bearing hosts contain humoral factors that can promote tumor growth and suppress immune responses.


Peripheral leukocyte migration inhibition reactivity to breast cancer antigens in patients with breast cancer and in normal controls. The peripheral leukocyte migration inhibition test has been used to assess cellular immunity to soluble antigen extracts of breast cancer in patients and normal controls. In sequential tests over several weeks, 23 of 23 patients with breast cancer in remission reacted intermittently, with 67 of 139 tests (48%) being positive (greater than or equal to 20% migration inhibition). Similarly, 6 of 10 patients in relapse reacted intermittently showing 16 of 61 positive tests (26%) and 126 of 129 normal females reacted intermittently showing 135 of 512 positive tests (26%). The mean percentage of migration inhibition for all tests in patients in remission was 16.4 +/- 1.2% and that for normal controls was 7.2 +/- 0.7%; this difference was highly significant (p less than 0.001). The value for all tests in patients with relapse was 11.8 +/- 1.4%; this was statistically lower than that for patients in remission (p less than 0.05) but statistically higher than that for normal controls (p less than 0.05). A few normal women, some with high risk factors such as a strong family history and/or fibrocystic and proliferative disease, had a mean percentage of migration inhibition value in the range of that for patients with breast cancer. Mean values of sequential tests may be a more meaningful index of cellular immunity against breast cancer antigen in all groups.


The reversible expression of an adult isozyme locus, Gdc-1, in tumors of the mouse. Tumors of the mouse possess 2 isozymic forms of L-glycerol-3-phosphate dehydrogenase (alpha-GPDH) (EC 1.1.1.8) that can be distinguished from each other by their heat inactivation and electrophoretic properties. These isozymes share certain structural features, since dissociation and reassociation of mixtures of the 2 isozymes lead to the generation of a hybrid molecular species. This finding suggests that the structural genes for these isozymes are closely related. A number of spontaneous and transplantable tumors of the mouse have been analyzed in order to assess whether the pattern of embryonic and adult alpha-GPDH isozyme expression is correlated with the degree of tumor differentiation. The results indicate that no correlation between the type of isozyme expressed and the degree of tumor differentiation or growth rate was evident. A striking correlation exists, however, between the physical form of the tumor and isozyme expression; all solid tumors possess, predominantly, the adult isozymic form of L-glycerol-3-phosphate dehydrogenase, whereas all ascites tumors, including embryoid bodies from ovarian and testicular teratomas, possess the embryonic form. A solid tumor, the C1300 neuroblastoma, that initially possessed the adult isozyme, was cultured in vitro; this resulted in the disappearance of the adult isozyme and predominant expression of the embryonic isozyme. Reinjection of cultured neuroblastoma cells into a host mouse produced a solid tumor that possessed the adult isozyme. The exclusive presence of either adult alpha-GPDH in solid tumor growths or embryonic alpha-GPDH in ascites tumor growths after converting from one physical forms of the tumor to the other, strongly supports a genetic regulatory mechanism which depends on the reversible repression and activation of the structural loci for these isozymes.


In vivo clonogenic tumor cell kinetics following 1,3-bis(2-chloroethyl)-1-nitrosourea brain tumor therapy. An in vitro colony formation assay was modified to determine the effects of in vivo 1,3-bis(2-chloroethyl)-1-nitrosourea therapy on tumor cell kill and subsequent clonogenic cell kinetics. The measured surviving fraction must be multiplied by the relative total number of tumor cells for each posttreatment interval in order to eliminate inaccuracies caused by dead cell removal in vivo and the lysis of damaged cells by the disaggregation procedure. The assumptions, limitations, and applications of the technique are discussed. 1,3-Bis(2-chloroethyl)-1-nitrosourea doses of 0.25, 0.50, and 1.00 X dose lethal to 10% of animals resulted in approximately at 1-, 2-, and 3-log cell kill, respectively. Significant proliferation of surviving clonogenic cells was observed after a latency period of approximately 2 days, and the rate of tumor regrowth was dose dependent. The cell-doubling times following treatment with 0.25, 0.50, and 1.00 X doses lethal to 10% of animals were 15, 21, and 38 hr, respectively. The interval to complete repopulation of the clonogenic pool corresponds to the observed increase in animal life-span for the 2 larger doses and further validates the assay as a true measure of in vivo chemotherapeutic efficacy.


Effects of grafts of single anterior pituitary glands on the incidence and type of mammary neoplasm in neutron- or gamma-irradiated Fischer female rats. Three batches comprised of 48 young adult Fischer female rats each were subjected to total-body irradiation with 50 rads modified fission neutrons, or were given 600 rads 137Cs gamma-rays, or served as unirradiated controls. On the day following exposure, one-half of each batch was grafted with a single anterior pituitary gland beneath the left kidney capsule. The animals were observed for mammary neoplasia and all those that died during the experiment were autopsied. The experiment was terminated 538 +/- 13 days after irradiation when all neutron-irradiated, pituitary-grafted animals had one or more mammary tumors. Only 2 of the 23 untreated rats that survived until termination of the experiment developed mammary fibroadenomas, and none had mammary carcinomas. The incidence of fibroadenomas was increased, and a single carcinoma was found, in unirradiated rats with pituitary grafts. Irradiation alone caused an increase in the incidence of mammary fibroadenomas and the appearance of carcinomas. Fibroadenomas were markedly increased by the addition of pituitary grafts to irradiation. Carcinoma incidence was less markedly affected. The neutron dose of 50 rads was slightly more effective in inducing mammary neoplasms than the 600-rad dose of gamma-rays.


Growth rates of normal and abnormal human mammary epithelia in cell culture. The in vitro growth rates of different classifications of human mammary epithelium were compared. Samples included 4 established breast cell lines and excised tissue or breast fluid cells originating WOMEN FOR 40 DIFFERENT AND comprising 3 classifications: normal, nonmalignant atypical, and malignant. Growth was quantitated in situ and expressed as population-doubling time. Principal findings were: (a) malignant cells divided at a slower mean rate than normal cells; (b) population-doubling time values of malignant cells were more heterogenous than those of normal cells; (c) cultures from nonmalignant atypias showed population-doubling time means and standard deviations between those of normal and malignant cells; and (d) long-term mammary tumor cell lines divided more slowly than did normal cells. Discussion includes implications of data for the preneoplastic state and cell culture of mammary epithelium.


Hyperplasia of the parathyroids and the pituitary in rats with experimental leydigiomas and inhibitory effect of several steroids. Bilateral orchidectomy performed on 10- to 15-day-old Wistar rats, immediately followed by the homograft of one testis into the splenopancreatic epiploon, resulted 6 months later in leydigiomas at the site of the graft in 100% of the animals. After this latency period, the tumors continued to grow proportionately to time elapsed and were associated with both pituitary microadenomas of the aldehyde-fuchsin-, aldehyde-thionin-periodic acid-Schiff-, and periodic acid-Schiff-positive cells and diffuse hyperplasia of the parathyroids, affecting in particular the chief cells of the latter. After a 6- or 12-month leydigioma latency period, the rats were given a daily treatment by gavage for 45 days of one or two of the following steroids: Compound A, 17alpha-ethynyl, (5alpha)-androst-2-en, 17beta-ol (17 beta-acetate) (5 mg/kg when alone; 2.5 mg/kg when combined); Compound B, 17 alpha-ethynyl, (5 alpha)-androst-2-en, 17 beta-ol (17 beta-nicotinate) (always given in combined form at 2 mg/kg); Compound C, 16-oximinoestrone, 3-allyl ether (2 mg/kg when combined). The treatments caused, in various degrees, (a) involution of the leydigiomas until complete disappearance in up to 50% of the cases; (b) recovery of the characteristic morphological pattern of the pituitaries; and (c) cytolysis and interstitial fibrosis in the previously hyperplastic parathyroids.


The effect of colchicine on the induction of ornithine decarboxylase by 12-O-tetradecanoyl-phorbol-13-acetate. The induction of mouse epidermal ornithine decarboxylase, 1 of the earliest and largest phenotypic changes following treatment of mouse skin with the tumor-promoting agent, 12-O-tetradecanoyl-phorbol-13-acetate, can be inhibited by prior administration of colchicine. Maximal inhibition of this enzyme induction was observed when colchicine was injected i.p. 90 or 120 min before promoter treatment, although time intervals up to 20 hr between colchicine and promoter treatment were effective. The effect of colchicine was dose dependent, with a dose as low as 25 nmoles/mouse causing an inhibition of 35%. Other microtubule-disrupting agents, vinblastine, vincristine, and Colcemid, had a similar effect on ornithine decarboxylase activity. However, beta, gamma-lumicolchicine, a photochemical derivative of colchicine with no antimitotic or microtubule-disrupting ability, and cytochalasin B, an inhibitor of microfilament-dependent processes, had no effect. N6, O2'-dibutyryl 3',5'-cyclic adenosine monophosphate, when administered just before colchicine, blocked the inhibitory action of colchicine. The results of these studies suggest that colchicine-sensitive structures, most likely containing microtubules, may be mediating elements between the binding of tumor promoters, perhaps to specific cell surface receptors, and the subsequent induction of ornithine decdaboxylase.


Action of the vinca alkaloids vincristine, vinblastine, and desacetyl vinblastine amide on microtubules in vitro. The Vinca alkaloids differ in their chemotherapeutic effectiveness and their toxicities. To determine whether differences are due to a differential effect on the assembly of tubulin into microtubules, we examined the effects of vincristine, vinblastine, and a newer alkaloid, desacetyl vinblastine amide, on the assembly of bovine brain tubulin in vitro. The three compounds block bovine tubulin polymerization in vitro and almost equally effectively at a 1 muM concentration (tubulin, 6.5 muM). At 10 muM, the three alkaloids also interact with preformed microtubules in vitro, causing spiral-like distortions of the microtubules. No effect of the alkaloids on polymerization of another fibrous protein, actin, was observed. Thus the differential actions of vinblastine, vincristine, and desacetyl vinblastine amide in vivo seems to be based on some biological process other than the reaction with tubulin or the microtubules per se.


Alterations in adriamycin efficacy by phenobarbital. Adriamycin dosage should be reduced in patients with impaired liver function, since adriamycin disposition is influenced by liver metabolism and biliary excretion. It follows that drugs that increase the metabolism or excretory capacity of the liver may decrease adriamycin concentrations to suboptimal values. Adriamycin metabolism was therefore studied in mice pretreated with phenobarbital (75 mg/kg i.v.) by injection. After an i.v. dose of adriamycin (30 mg/kg i.v.), plasma fluorescence due to drug and metabolites was less and disappeared at a greater rate in phenobarbital-pretreated mice than control animals. When extracted with chloroform: isoprophyl alcohol (1:1), the livers from the phenobarbital-pretreated group yielded a greater concentration of glycones. Experiments with liver microsomes confirmed that aglycone production occurred at a more rapid initial rate in phenobarbital-induced livers. No increase in aldoketo reductase (daunorubicin reductase) activity was noted. Phenobarbital-pretreated mice, inoculated i.p. with 1 million L1210 cells and then treated with adriamycin (6 mg/kg i.v.), had significantly lower survival than did controls (p less than 0.01). These findings show that phenobarbital affects the disposition of adriamycin by microsomal enzyme induction and suggest that drugs that induce microsomal enzymes should not be used concurrently with adriamycin if optimal drug efficacy is desired.


Fetal calf serum-induced blastogenic and cytotoxic responses of human lymphocytes. Incubation of normal human peripheral blood lymphoid cells in fetal calf serum-supplemented media initiates blastogenic and nonspecific cytotoxic responses. A panel of lymphocytes from 13 normal donors exhibited increased [3H]thymidine incorporation from 2 to 40 times that of control lymphocytes incubated in pooled human AB-supplemented media. Fetal calf serum-sensitized human lymphoid cells were active in cytotoxicity assays against a wide variety of cultured human tumor and normal target cells. Cytotoxicity continued to increase from 3 to 9 days of incubation in fetal calf serum-supplemented media, while blastogenic response peaked at 6 days of incubation. Cytotoxic and blastogenic responses increased with higher concentrations of fetal calf serum. Once stimulated with fetal calf serum, the lymphocytes did not require the continued presence of fetal calf serum to mediate a cytotoxic reaction.


A viral immunodepressive factor associated with experimental mouse tumors. An immunodepressive factor (IDF) capable of inhibiting the rejection of allogeneic lymphomas in mice was detected in a number of transplantable mouse tumors. The allograft reactivity of the host was impaired for at least 10 months after IDF administration. No clinical evidence of bacterial or viral infection was detected in IDF-treated animals. Sera collected from mice at various intervals after injection of IDF showed a great increase in the IDF-titer. The IDF persisted in mouse sera for at least 3 months, whereas no IDF activity was found in sera of rats given injections of the factor. IDF was capable of replicating in vitro in mouse embryo cells, but not in rat embryo or HeLa cell cultures. IDF was inactivated in vitro by heat (65 degrees for 30 min), ultraviolet light or ether, but not by ethyl alcohol. These studies indicate that IDF is a virus capable of producing a long-lasting asymptomatic infection specifically interfering with the host's allograft reactivity. In several instances a close association between the lactic dehydrogenase virus and IDF was found. Nevertheless no conclusion was reached on the identity or nonidentity between the two viruses.


Chemistry and structure-activity studies of the nitrosoureas. The N-nitrosoureas are the most active of a variety of N-nitroso compounds tested against L1210 leukemia in mice. Structure-activity studies show that the N-(2-chloro(or fluoro)ethyl)-N-nitrosoureido grouping is necessary for high-level activity in this test system, but a variety of carrier groups can be attached to the N'-nitrogen of the urea. For high-level activity against solid tumors in rodents, eg, the Lewis lung adenocarcinoma, the N'-substituent must be a cyclohexane ring, and the most active of these compounds are substituted at position 4 of the ring. The chemistry involved in the synthesis and reactions of the nitrosoureas is described. The possible relationship of the chemically reactive species generated by the decomposition of the nitrosoureas under in vivo conditions to biologic activity is discussed.


Randomized prospective trial comparing 5-fluorouracil (NSC-19893) to 5-fluorouracil and methyl-CCNU (NSC-95441) in advanced gastrointestinal cancer. The Southwest Oncology Group has confirmed the superiority of 5-fluorouracil (5-FU) and methyl-CCNU versus 5-FU weekly as treatment for patients with advanced gastrointestinal cancers. The drug combination produced a PR rate of 29.3% compared to the single-drug arm rate of 10.6%; however, the two-drug arm produced much more toxicity. Despite the improved response rate no improvement in survival was observed.


Acute changes in high energy phosphates, nucleotide derivatives, and contractile force in ischaemic and nonischaemic canine myocardium following coronary occlusion. Studies of ischaemic myocardium following occlusion of coronary arterial branches revealed a substantial decline in creatine phosphate, adenosine triphosphate, and total adenine nucleotide contents over 40 min. Contents of nucleotide breakdown derivatives increased significantly but transiently. Contractile force declined rapidly and stabilized at a low level. In nonischaemic muscle following occlusion creatine phosphate, adenosine triphosphate, and total nucleotide contents declined rapidly, although not to the degree seen in ischaemic muscle. Contents of derivatives increased only slightly. Contractile force increased.


An excitatory nociceptive cardiac reflex elicited by bradykinin and potentiated by prostaglandins and myocardial ischaemia. Application of bradykinin to the exposed ventricular surface of the dog heart elicits a reflex cardiovascular response which includes a rise in blood pressure, tachycardia, renal vasoconstriction and muscular vasodilation. The reflex response depends on the dose of bradykinin and is increased by concomitant application of prostaglandin E1 or E2 and reduced by indomethacin. Temporary occlusion of the coronary artery supplying the area of the ventricle under study also sensitized the heart to topical application of bradykinin. Bradykinin and prostaglandins are released by the heart during ischaemia. We suggest, therefore, that bradykinin and prostaglandins acting in concert are the natural stimulus for excitation of the sensory receptors signalling the pain of myocardial ischaemia. We also suggest that the nervous reflex which arises from activation of sympathetic sensory nerve endings is the mechanism subserving the cardiovascular events which accompany anginal attacks.


Effect of heart rate on zonal tension and ischaemia following coronary occlusion: optimal rate for Treppe versus ischaemia. The optimal heart rate in the immediate period following acute coronary occlusion has been controversial from the standpoint of arrhythmias and the extent of ischaemic injury. In the present study we have examined the effect of heart rate on both local myocardial contractile ability and ischaemia in 10 open chested dogs using strain gauge arches and epicardial electrograms. After sinus node destruction, atrial pacing was instituted for rate control at 100/min and increased randomly to 150, 175, and 200/min. Before coronary occlusion, total tension and rate of tension rise progressively increased at higher rates while ST segments demonstrated no significant changes. After coronary artery occlusion, total tension and rate of tension rise in the ischaemic zone decreased markedly and showed no significant change with increments in pacing rate. In the border zone, after the initial decrease in tension, pacing at 150 beats/min improved tension without a change in ST segments. However, when the rate was increased to 175 and 200 beats/min, although border zone tension increased further, ST segments rose significantly. Thus, a heart rate between 100-150/min provides the optimal range for increasing contractile ability in the nonischaemic and border zones without adversely affecting the degree of ischaemic injury.


Effect of hypercapnia and hypocapnia on myocardial blood flow and performance in anaesthetized dogs. The effects of hypercapnia and hypocapnia on haemodynamics, coronary blood flow, and lactate metabolism were evaluated in anaesthetized closed chest dogs. Coronary flow increased with increased pCO2 and oxygen consumption and left ventricular performance were well maintained. Hypocapnia reduced coronary flow, oxygen consumption, and left ventricular functional performance.


Effect of methylprednisolone sodium succinate on hypoxic heart muscle. The ability of 30 mg/litre methylprednisolone sodium succinate (MPSS) to modify the effects of hypoxia on isolated Langendorff-perfused rat hearts was investigated. When perfused under hypoxic conditions (pO2 less than 0.8 kPa[6 mmHg]) these hearts lose intracellular enzymes, including creatine phosphokinase (CPK) and succinic dehydrogenase (SDH). The size of the extracellular space is enhanced, the cells gain Na+ and Ca2+ and lose K+, and the endogenous stores of ATP and CP are depleted. Initially the resistance to flow in the coronary circulation falls but after 75 min of hypoxic perfusion it increases so that coronary flow is reduced. MPSS failed to prevent hypoxic muscle from either gaining Na+ and Ca2+ or losing K+. It did, however, delay the release of CPK and SDH from the hypoxic muscle, prolong the phase of increased coronary flow, and decrease the rate of depletion of the energy-rich phosphate stores. MPSS potentiated the hypoxic-induced gain in Ca2+. Whilst the effects of MPSS on coronary flow and tissue Ca2+ were probably due to the steroid part of the complex, the other changes, including the protection of the ATP and CP stores and the delayed enzyme release, were probably due to the presence of the sodium succinate.


Influence of training on myocardial responses of rats subjected to conditions of ischaemia and hypoxia. Physical training has been advocated to minimize the problems associated with coronary heart disease; however, the responsible mechanisms are obscure. Rats trained to run on a treadmill were subjected to acute conditions of hypoxia and myocardial ischaemia. Trained rats were better able to maintain a higher level of cardiac performance (dP/dt max) after hypoxia than non-trained rats, but no advantages were apparent after ischaemia. Biochemical data showed no myocardial differences between the groups in oxygen utilization or energy availability.


Influence of hyperthyroidism on glycerol-extracted cardiac muscle from rabbits. The mechanism responsible for the enhancement of myocardial contractility in hyperthyroidism is unclear. The possibility that this mechanism may involve a direct effect on the contractile proteins was investigated using the glycerol-extracted muscle strip from right ventricular papillary muscles of euthyroid rabbits and rabbits made hyperthyroid by the intraperitoneal injection of 0.25 mg/kg 1-thyroxine for 10 d. Intact papillary muscles from the hyperthyroid rabbits had an enhanced rate of tension development, a decreased time to peak tension, and a slight though insignificant increase in active tension compared to control animals. Maximal isometric contractions were induced in glycerol-extracted cardiac muscle strips from the two animal groups by the addition of 5 mmol/litre ATP and 5 mmol/litre MgCl in a buffer solution containing 0.15 mol/litre Tris-HCl (pH 7.1) at 26 degrees C. Peak isometric tension was increased in glycerinated muscle strips from hyperthyroid rabbits (1.52+/-0.10 vs 1.26 +/-0.13g/mm2), but the differences did not reach statistical significance. However, there was a marked increase in the rate of tension development in the hyperthyroid group (62.5+/-5.4 vs 41.8+/-4.7 mg/mm-2/s, P less than 0.01). This increase in the rate of isometric tension development in both intact and glycerinated muscles from hyperthyroid rabbits may be related to changes in the intrinsic turnover of actomyosin cross-bridge links in this condition. Thus, these findings suggest that thyroid hormone may influence cardiac muscle function by a direct effect on the contractile proteins.


Coronary occlusion before, during, and after strenuous exercise. Groups of dogs were exercised immediately before, and immediately after occlusion of the left anterior descending coronary artery. Coronary occlusion by a staged procedure resulted in a low mortality and rare ventricular ectopic beats. The combination of simultaneous exercise with coronary occlusion reproducibly provoked ventricular fibrillation (VF). Exercise before and after occlusion frequently produced ventricular tachycardia but no VF. The risk of major arrhythmias was related to the peak heart rate resulting from the exercise.


Tissue culture, protein and collagen synthesis in antibiotic sterilized canine heart valves. Viability of canine heart valve leaflet fibroblasts was assessed after varying periods of sterilization and storage in antibiotic-nutrient solution. Tissue culture and assessment of protein and collagen synthesis showed that tissue obtained under optimal conditions rarely retains viability beyond 3 weeks in antibiotic-nutrient solution and is severely impaired after 2 weeks. This casts serious doubts on viability in current clinical homograft valve practice.


Viability in human heart valves prepared for grafting. Viability of antibiotic sterilized and stored human heart valves obtained at routine necropsy was assessed by tissue culture and protein and collagen synthesis. Only three of 23 valves examined showed any evidence of viability, in striking contrast to earlier work on canine valves obtained under optimal conditions. These findings justify doubts regarding pre-implantation viability in human heart valves prepared for grafting.


Continuous imaging of regional myocardial blood flow in dogs using krypton-81m. The unique physical properties of the freely diffusible gas krypton-81 m allowed continuous imaging of regional myocardial blood flow in dogs when infused into the aortic root. Regional changes in myocardial perfusion related to transient coronary artery occlusion were demonstrated both as high resolution gamma camera images and as a quantitative strip chart record.


Further studies of the epithelium covering preovulatory rabbit follicles with special reference to lysosomal alterations. The fine structure of the surface epithelium over preovulatory rabbit follicles was examined parallel with visualization of acid phosphatase at the electron microscopical level. Small enzyme positive vesicles were pinched off from the Golgi cisternae and similar vesicles fused and got incorporated into larger lysosomes of dense body type. Some lysosomes appeared in direct continuity with tubular enzyme positive structures. Other possible ways of increase of the lysosomal pool are indicated and discussed. As in previous studies a maximal accumulation of lysosomes was found in the apical epithelium at 8 h after an ovulatory dose of human chorionic gonadotrophin (HCG); thereafter a gradual loss of lysosomes ensued. Before the lysosomes disappeared from the surface epithelium they changed in character. They became more electron-lucent and revealed a fine-fibrillar matrix. Dense bodies deep in the cell interior appeared to communicate with each other and with the extracellular space below the surface epithelium. Openings were never seen towards the peritoneal cavity. tthe loss of lysosomal content from the apical surface epithelium before follicle rupture appeared in many respects similar to the histamine release process in mast cells. tthe findings support our working hypothesis that the surface epithelium over tgraafian follicles is an essential source of proteolytic enzymes and that these may be released extracellularly and actively contribute to the dissolution of the follicular apex before rupture.


Developmental changes in synaptic formation in the hypothalamic arcuate nucleus of female rats. The hypothalamic arcuate nucleus (ARCN) of female rats at 5, 20, 45 and 90 days of age was examined ultrastructurally. Axodendritic and axosomatic synapses were counted in 18,000 mum2 area of the ARCN in each brain. Axodendritic and axosomatic synapses in the ARCN of day 5 rats were very small in number. Axon terminals contained small spherical vesicles (SSVs, 40-60 nm in diameter). Occasionally large granular vesicles (LGVs, 75-130 nm in diameter) were found to coexist with SSVs in the endings. Pre- and postsynaptic membranes were thin. The ARCN at this age exhibited a large extracellular space which decreased with advancing age. In day 20 rats, axodendritic and axosomatic synapses increased in number up to about one-half of those of day 45 or day 90 animals. Synaptic vesicles increased in number and mitochondria were frequently encountered in the axon terminals. Pre- and postsynaptic membranes became thicker than those of day 5 rats. Further increase in the number of axodendritic and axosomatic synapses in the ARCN of day 45 rats was observed, and there were no significant difference in the morphology and incidence of synapses between day 45 and day 90 rats. Synaptic vesicles were numerous and pre- and postsynaptic membranes were thick. In tissue incubated with 5-hydroxydopamine (5-OH-DA) before fixation, small granular vesicles (SGVs, about 50 nm in diameter) which were labeled with 5-OH-DA were detected in a certain number of endings in all material taken from each age group, but the incidence of synapses containing SGVs was usually low. From these results, it can be proposed that an increase in the number of synapses in the ARCN is correlated wihh functional maturation of the ARC neurons.


Studies with mature rat hepatocytes in monolayer culture. I. Propagation of isolated hepatocytes, morphological and biochemical in culture. Differentiated cells have been established in monolayer culture from adult rat liver and their ultrastructural and biochemical features characterized after 20-30 generations. Hepatocytes were isolated by enzyme perfusion of the liver followed by treatment with papain, which allowed cultures to be established more readily and to be cloned at an early stage. Ultrastructural studies indicated that the cells were derived largely from hepatic parenchymal cells. The cells showed structural modifications during primary culture but were stable thereafter. The cultured cells retained some differentiated functions unique to liver cells, including the synthesis of ornithine form arginine and the secretion of serum proteins, albumin, chi- and beta-globulins.


The effects of starvation on the planarian worm Polycelis tenuis Iijima. Employing a combination of microscopical, biochemical and autoradiographic techniques, the primary effects of starvation on adult polycelis tenuis have been studied. Over a five week period of starvation there is on average a 32% decrease in the size of the organism. This decrease is contributed to by a reduction in mitosis and an increase in cell shrinkage autolysis and death. During starvation (following a sharp rise in RNA synthesis) there is a distinct sequence of events; four peaks of acid phosphatase activity can be resolved. The first is associated with the immediate response of the gastrodermis to feeding; the second (after 6 to 7 days) with increased autophagy and dedifferentiation in the gland cells and with muscle lysis of cells. The third peak (after 14 to 15 days) is contributed to largely by the lysis of cells in the gut and the fourth peak (after 25 to 26 days) is caused by an extensive lysis of the reproductive system. Fine structural changes involving increased intracellular vacuolation, autophagy, crinophagy, atrophy of muscle, increased intercellular space and loss of basement membrane matrix have been related to changes in enzyme pattern. Nerve cells appear unchanged throughout the first five weeks of starvation. Pigment and gland cells loose their characteristic granules, dedifferentiate and become morphologically similar to the undifferentiatied neoblasts. Dedifferentiation and the mechanisms involved in the survival of starvation are discussed.


Regional distribution of three ultrastructural retinula types in the retina of Cataglyphis bicolor Fabr. (Formicidae, Hymenoptera). The retina of Cataglyphis bicolor was investigated by electron microscopy. Three types of structurally distinct retinulae were found and mapped throughout the compound eye: Type I is composed of four unpigmented thin cells, four larger pigmented cells as well as a basal ninth cell. Its rhabdom possesses a round cross section and four microvilli directions. This type occupies most of the dorsal two-thirds of the retina. Type II consists of two thin cells, two intermediate cells and four large cells. A basal ninth cell is also present; the rhabdom is as in type I. Type II retinulae are located in the ventral third of the retina. Type III ommatidia are unique within the Hymenoptera: there are four large pigmented cells, four thinner unpigmented cells and a basal ninth cell. The rhabdom, however, has a dumb-bell shaped cross section; two small cells lie at its opposed extremities and the remaining six cells have mutually perpendicular microvilli orientations. This type of retinula is found at the dorso-medial eye margin. Serial sectioning in this region revealed a conical shaped rhabdom without any torsion along the longitudinal axis. The rhabdomere cross section was calculated from distal and proximal thin sections. Angular statistics were applied to the microvilli directions of all three ommatidial types to determine the degree of order. A possible functional significance  of the structural specializations of the different eye regions is discussed.


The fine structure of the carapace integument of Daphnia magna Straus (Crustacea Branchiopoda). The structure of the two integumental layers comprising the carapace of female D. magna was examined at several points through the molt cycle. The epicuticle and procuticle are simple in organisation; pore canalsare absent but intracuticular fibres are present, forming complexes with invaginations of the epidermal plasma membrane similar to such complexes described in the literature for othe arthropods. The epidermis consist almost entirely of cuticle-secreting cells. Secretion of the new cuticle begins when 50-67% of the instar has elapsed by which time the epidermal cells have increased in height and their nuclei have become more rounded. However, other presumed secretory phenomena observed viz. the formation of dense core vesicles by Golgi bodies, and the occurrence of these and coated vesicles near the apical plasma membrane are not restricted to any particular period during the molt cycle. This suggests that the mechanisms of cuticle secretion do not undergo marked changes in activity as they do in decapods; presumably this relative continunity is related to the much shorter molt cycle of cladocerans.


Scanning electron microscopic studies on the development of the chick retina. Fixed retinae of chick embryos and chicks of the first week after hatching were fractured and examined with the scanning electron microscope. The matrix cells of the retina proliferate up to the beginning of the second week. The migrating cells are oriented in cell cords. This columnar organizaion prevails up to the development of the plexiform layers formed as a consequence of the outgrowth of the dendritic and axonal cell processes. Special attention was paid to the differentiation of the ganglion, bipolar and receptor cells, and the radial fibers (Müller cells). Two main morphological patterns are significant for the organization of the retina during neurogenesis: a)the cell to cell contacts of migrating cells and b)the spatial arrangement of Müller cells which could provide guidelines for migration of neuronal elements.


Quantitative morphological study of smooth muscle cells of the guinea-pig taenia coli. A quantitative study of muscle cells of the guinea-pig taenia coli is reported. Stereological methods were used on electron micrographs and phase contrast micrographs. Smooth muscle cells of taeniae fixed under 1 gram load were about 515 mum long. Muscle cell volume was about 3,500 mum3 and cell surface 5,300 mum2. About 168,000 caveolae were found at the surface of each muscle cell, covering about 29% of its surface. They produced a 73% increase of the cell membrane compared to a smooth-surfaced cell. The ratio surface-to-volume is about 1:0.67 if the geometrical surface is considered, or 1:0.39 if the total surface of the cell membrane (including the caveolae) is considered. Mitochondria constituted 3.5-4% of the cell volume. A few nexuses were observed, both between two muscle cells and between a muscle cell and an interstitial cell. In serial sections septa of connective tissue and groups of muscle cells were found to disappear within few tens of microns or to merge with other septa, and the taenia did not appear to be divided into clear-cut muscle cell bundles. Bundles of smooth muscle cells were seen passing from the taenia to the underlying circular muscle. The transverse sectional area of the taenia ranged between 0.14 and 0.39 mm2; it showed about 526 blood vessels-mm-2.


Structural changes in smooth muscle cells during isotonic contraction. Smooth muscle cells of the guinea-pig taenia coli were studied in light and electron microscopy, in condition of mild stretch or of isotonic contraction. During contraction the cells increase in transverse sectional area and their packing density passes from 94,000-mm-2 to 18,000-mm-2. The percentage increase in transverse sectional area of the taenia is approximately the same as the percentage decrease in length. Measurements of cell transverse sectional area suggest that the individual cells shorten and fatten more than the taenia as a whole. Whereas stretched muscle cells run parallel to each other and show a fairly smooth surface, isotonically contracted cells are twisted and entwine around each other. Their surfaces are covered with myriad processes and folds. Longitudinal, transverse or oblique stripes are seen in light microscopy in the contracted muscle cells and it is suggested that they are related to the characteristics of the cell surface. In electron microscopy a complex pattern of interdigitating finger-like and laminar processes is observed. Caveolae are mainly found on the evaginated parts of the cell surface, dense patches are mainly (but not always) found on the invaginated parts. Desmosome-like attachments between contracted cells are frequent. The collagen fibrils run approximately parallel to the stretched muscle cells; on the other hand, they run obliquely and transversely around the isotonically contracted cells.


Studies on intracellular transport of secretory proteins in the rat exocrine pancreas. V. Kinetic studies on accelerated transport following caerulein infusion in vivo. The previous finding that intracellular transport of secretory proteins in the rat exocrine pancreas is accelerated by in vivo stimulation with a pancreatic secretagogue has been further analyzed. Using a radioassay for discharge of newly synthesized proteins, the rate of release was compared in control and prestimulated lobules. In control preparations discharge occurred with an initial lag period of 30 minutes and a maximum after two hours of incubation. After in vivo infusion of 5 x 10(-8) g/hr. caerulein for 24 h in vitro discharge started after 10 minutes of in vitro incubation and attained a maximal rate after one hour. Using the same radioassay and several inhibitors of intracellular transport and granule discharge, it could be demonstrated that both processes were reduced to the same extent in controls and in lobules with accelerated transport. To obtain direct evidence for the degree of acceleration of the different transport steps between rough endoplasmic reticulum, Golgi complex and zymogen granules, the respective subcellular fractions of these organelles prepared and characterized ultrastructurally and biochemically. The rate of disappearance of newly formed proteins from rough microsomes and the appearance in smooth microsomes and zymogen granules were significantly increased after in vivo stimulation. The data substantiate an acceleration of the regular transport steps by the secretagogue. There was no indication that a high level of secretory activity leads to a rerouting of secretory proteins or to an omission of one of the regular steps in intracellular transport.


Sieve structure of slit diaphragms of podocytes and pore cells of gastropod molluscs. The ultrastructure of the slit diaphragms between the pedicels of the podocytes of the prosobranch Viviparus viviparus and between the cytoplasmic tongues of the haemocyanin producing pore cells of the pulmonate Lymnaea stagnalis was investigated. In both cell types 2 diaphragms are present in the slits. They form a 3-dimensional sieve structure with holes of respectively 90 x 110 A (podocyte) and 200 x 220 A (pore cell). Injection experiments showed that the size of the holes of the pore cell sieve matches that of particles which can be ingested by theis cell type. The substructure of the sieves of the molluscs is compared to that of the 2-dimensional sieve of the podocytes of the mouse and the rat.


Monoaminergic neurons in the nervous system of crustaceans. Certain neurons in the nervous system of the malacostracan crustaceans give rise to a predominantly green and a sparse yellow fluorophore in the histochemical fluorescence method of Falck-Hillarp. The same applies to the whole of Crustacea. The green fluorophore is probably a catecholamine; the yellow to brown-yellow has not yet been identified. The biogenic amine responsible for the green fluorescence, besides being found in diffusely distributed fibres, also appears in distinct areas of fibre concentrations in the central nervous system. The protocerebrum of the malacostracans contains three areas: the central body and two areas in the top of the brain, one anterior and one posterior. The latter two are not recognized as separate areas in ordinary histological preparations. In addition, the optic neuropiles are fluorescent, some with a distinct stratification of the fluorophore. The deuto- and tritocerebrum and the ventral nerve cord also contain monoaminergic neurons. Of the brightly fluorescent areas in the whole of Crustacea, only the central body consistently exists in all species. The other areas of concentrated fluorescent neuropile are restricted to smaller taxonomic units and differ from each other.


Some features of the ultrastructure of the coenecium of Rhabdopleura compacta. The coenecium of Rhabdopleura consists of a series of tubes, some erect and some repent. These tubes are composed of rings, one stacked within another. The rings are smooth on the inside surface and rough outside. Newly laid down rings are thin and smooth on both surfaces, fibrous material is laid down on the external surface during growth in thickness by the cephalic shield of the zooid. The erect tubes remain discrete, but the repent tubes, which are attached to the substratum can become incorporated in a mass of secreted material. The external vertical fibres cross several rings and probably serve to anchor the stack. Besides these fibrils that run for several segments, there are other shorter fibres that run along the length of each cylindrical ring, and are not continuous across the rings. These long and short fibres have features in common with those found in the graptolites.


Digestive enzyme secretion in Stomoxys calcitrans (diptera: muscidae). Enzyme assays and morphological and histological studies show that the opaque zone midgut cells of the hematophagous fly Stomoxys calcitrans are responsible for the production of proteolytic digestive enzymes and that these are secreted into the gut lumen via membrane bound vesicles (MBV). The secretory cycle can be summarized as follows: initially the rough endoplasmic reticulum is stacked and the apices of the cells are packed with MBV. This is followed by a period of release characterized first by cytoplasmic extrusions containing high densities of MBV, then by microvesiculation of the microvilli combined with a progressive distribution of rough endoplasmic reticulum and lightening of the cellular cytoplasm. Glycogen appears in the cells at this stage and is gradually lost as the rough endoplasmic reticulum becomes stacked once more and the numbers of MBV build up again. The cycle which occurs regularly and synchronously in the cells of the zone repeats itself many times up to the completion of digestion of the blood meal. The secretory cycle is discussed with reference to activity in other secretory tissues.


Mast cell activation and tissue cell proliferation. The effect of mast cell activation and degranulation on the proliferation in the intact mesentery was studied in Sprague-Dawley rats. Mast cell activation was achieved by a single intraperitoneal injection of Compound 48/80. The proliferation was studied using three independent methods for estimation of cell production and DNA synthesis: 1. the mitotic index, 2. the relative number of cells having a DNA content in the S and G2 regions, by Feulgen photometric measurement in individual cells, and 3. the specific DNA activity, employing a method which combines a liquid scintillation technique after an intravenous injection of 3H-thymidine and Feulgen photometric determination of the DNA content per membrane preparation. It was found that the proliferation of the normal mesenchymal cells adjacent to the activated and degranulated mast cells in the mesentery was significantly increased within 24 and 32 h, the maximum increase being more than 20-fold compared to untreated controls. The results suggest that the common type of mast cell may have a pathophysiological function related to stimulation of local cell proliferation.


Ovarian follicles of normal NMRI mice and homozygous "nude" mice. 1. Quantitative methodological study in the pubescent "nude" mouse. It is possible to determine statistically the repartitions of shapes or sizes in a population of particles from the recurrence of shapes or sizes of their outlines in microscopical sections. This method was applied to a population of 1326 follicular profiles randomly sampled from 30 ovaries of pubescent homozygous "Nude" mice. Under experimental conditions, the follicles were not spherical but could be assimilated to prolate ellipsoids with a mean eccentricity of 0.81. The average radius of the follicles taken as a whole was 20.2+/-0.7 (S.E.) mum. From these, the average radius of only the non-primary follicles was 58+/-1.3 (S.E.) mum. The volumetric repartition of the ovarian follicles indicated that 62% of the total follicular mass was made of follicles with a radius greater than 115 mum, although these represent only 2% of the total number of follicles.


Ultrastructure of the membrane attachment sites of the extrusomes of Ciliophrys marina and Heterophrys marina (Actinopoda). The actinopods Ciliophrys marina and Heterophrys marina both have membrane bounded extrusomes attached to their cellular and axopodial membranes. The extrusomes of C. marina, the muciferous bodies, are fairly simple in structure and contain a homogeneous osmiophilic substance. Their attachment site is characterized by a rectangular array of freeze fracture particles in the cell membrane. The extrusomes of H. marina, the conicysts, are more complex and contain a two-part osmiophilic body. The attachment site of conicysts is characterized by a rosette of 8 freeze fracture particles very similar to the 9-particle rosette found at the mucocyst attachment sites in Tetrahymena. Furthermore, intracytoplasmic bridges connect the conicyst and cell membrane faces, and a specialized fibrillar structure is found on the cell membrane in the region of conicyst attachment. The various possible roles for such particle arrays are discussed and their presence in virtually all extrusomes is predicted.


Enveloping layer and periderm of the trout embryo (Salmo trutta fario L.)20U. The origin of pericarderm and epidermis has been studied in trout embryos from stage 20(2 days after fertilization at 10degrees c) to hatching (stage 410). Between stages 20 and 50, the blastodisc consists of an inner mass of blastomeres covered by a superficial layer of closely packed blastomeres, the peripheral layer. This layer gives rise to both peripheral cells and to cells joining the inner mass of blastomeres. Between stages 50 and 110, junctions differentiate between peripheral cells. This newly formed superficial epithelium the enveloping layer, no longer gives rise to inward migrating cells. From stage 110 on, a basement membrane differentiates beneath a one-cell thick subperipheral layer, which thus becomes the ectodermal basal layer, the prospective epidermal basal layer. From these and ultrastructural observations, it is concluded that 1)the epidermis apparently originates, at least in part from the peripheral cells, between stages 20 and 50; 2) the periderm assumes a protective function over the body of the embryo and also a secretory function over the yolk sac (probably producing the hatching enzymes); 3)the periderm, which is a temporary structure, appears to play the role of an embryonic membrane in teleosts.


The Wolff-Parkinson-White syndrome: the value of the HIS bundle electrogram,. The HIS bundle electrogram has led to the aquisition of additional information on the physiology of the WPW syndrome and has become a useful technique for its diagnosis. The findings on the HBE and their interpretation in WPW are reviewed.


Right ventricular mass estimation by angioechocardiography. A combined angiocardiographic-echocardiographic method for estimating right ventricular wall mass is described. Biplane cineangiocardiograms are analyzed for ventricular volume in end-diastole, and wall thickness is determined from echocardiograms obtained with a high frequency transducer and strip chart recorder, The intracavitary and the external surface volumes of the ventricle are derived, and the difference multiplied by 1.050, the specific gravity of myocardium. Excellent correlation was observed between right ventricular wall mass and body surface area in normal children (r = 0.93). The mean right ventricular mass was 44.5 g/M2 as compared to 78.1 g/M2 for the left ventricle, corresponding mass/EDV values were 0.48 g/cm3 and 1.26 g/cm3, respectively. In isolated right ventricular pressure overload, the increase in right ventricular mass is chiefly due to the increase in wall thickness; in volume overload, it is due mostly to the increase in chamber volume,


Cine angiographic diagnosis of patent foramen ovale in paradoxical embolism,. A case of paradoxical embolism is presented in which the antemortem diagnosis was facilitated by a new simple angiographic technique that demonstrated a patent foramen ovale. An additional 14 patients were studied by this technique, and one had a patent foramen ovale. The mechanism of reversal of the normal intraatrial pressure gradient and subsequent right to left shunt through a patent foramen ovale is discussed. This angiographic technique may be readily applied in any diagnostic catheterization laboratory and complements existing methods for detecting patency of the foramen ovale in patients with suspected paradoxidal embolism.


The evaluation of routine selective arteriography in the investigation of renovascular hypertension. A comparison of lesions discovered by selective renal arteriograms with abnormalities observed on abdominal aortograms was made in 100 consecutive hypertensive patients. Twenty-one lesions not seen on aortograms were found by selection. Eight patients with a normal aortogram had lesions. An abnormality was discovered in at least one study in 58 patients. The increased rate of lesion detection by selective studies outweighs the slight risk of complications.


Aortic stenosis, angina pectoris, coronary artery disease. The data from 88 patients (pts) with aortic stenosis (AS) were reviewed to determine relationships between angina pectoris (AP) and coronary artery disease (CAD). Results of surgery performed in 81 of these pts was analyzed. All pts had coronary arteriograms, and lesions greater than or equal to 50% were considered significant. Fifty-nine pts had an aortic valve gradient measured at catheterization greater than or equal to 40 mmHg, and in 29 pts, AS was confirmed at operation. Sixty-eight pts (77%) experienced AP, and 32 had coexisting CAD (47%); 9 of 20 pts without AP had CAD (45%). There were no significant differences in the incidence of AP in pts divided into subgroups by the aortic valve gradient (40-50, 51-100, 101-200 mmHg) or age (40-59, 60-81 years). Also, no significant differences were found in the incidence or extent of CAD between the two age groups; the extent of CAD was similar regardless of the presence or absence of AP. In pts with AP (1) CAD was more likely in pts greater than or equal to 60 years of age; (2) CAD was less likely when the aortic valve gradient was greater than 100 mmHg, suggesting that AP in these pts was due to hemodynamically severe AS. All pts with 3-vessel CAD experienced AP, and the aortic valve gradient was less in these pts than in those with no CAD or less extensive CAD. In 19 pts with combined AS and CAD who had both the aortic valve replaced and a revascularization operation only 1 of pts died in the hospital, while 3 of 19 pts with combined AS and CAD who had aortic valve replacement alone died. In this study a significant number of pts with AS experienced AP, and the presence or absence of AP did not predict coexisting CAD. Coronary arteriography is recommended in the evaluation of pts greater than or equal to 40 years of age with AS. The operative mortality appears to be decreased in pts with AS and CAD who have combined surgery.


Ejection time--heart rate relationship during exercise. The slope of the regression equation for left ventricular ejection time (LVET) vs heart rate (HR) is the appropriate factor for correcting LVET for HR. Because the regression relationship varies under different conditions, we determined the LVET-HR equations for subjects (1) seated at rest on a bicycle ergometer, and (2) during uninterrupted bicycle exercise. In 18 normally active male volunteers, ages 22-37, HR and LVET were measured under the two conditions and the regression relationship for LVET on HR determined for each. Regression equations are as follows: (1) LVET = 379-1.8 HR +/- 11.0 for subjects seated at rest on a bicycle ergometer, and (2) LVET = 371-1.2 HR +/- 13.9 for subjects performing upright bicycle exercise. The slope factors (1.8 and 1.2) differed significantly (p less than 0.01). The data indicate that considerable error can result from arbitrarily applying to exercising or resting subjects a correction factor which does not fit the conditions of the data to be corrected.


Potential artifact in measurement of left ventricular filling pressure with flow-directed catheters. Artifactual pulmonary wedge pressure measurements were encountered during bedside use of Swan-Ganz catheters. These values were higher than pulmonary artery end diastolic pressure and devoid of typical phasic contours. Utilizing fluoroscopy these artifacts were reproduced by advancing the catheter tip 2-4 cm beyond the site of initial wedging and reinflating the balloon. Spurious increases of pulmonary wedge pressure of up to 15 mm Hg were thus obtained. In vitro catheter testing demonstrated overlapping of the catheter tip by the deformed balloon when it was fully inflated in a channel too small to accomodate it. These measurement artifacts are thus attributed to distal migration of the catheter tip into relatively small pulmonary artery branches and to subsequent occlusion of the catheter lumen by the balloon when it is reinflated. This can readily be avoided by routinely inflating the balloon with the minimum volume of air sufficient to yield a pulmonary wedge pressure tracing.


A new wedge to facilitate obtaining half-axial views during coronary arteriography. A wedge has been designed and constructed to facilitate obtaining posteroanterior and oblique half-axial projections routinely during coronary arteriographic examinations in a conventionally equipped catheterization laboratory.


A semiautomated technique for the rapid evaluation of left ventricular regional wall motion. A semiautomated system for the rapid evaluation of left ventricular regional wall motion is described. The system includes direct projection of the cineangiogram film on an X-Y digitizer which is interfaced with a PDP-9 computer. The ventricular model used to evaluate regional wall motion and the methods of data analysis are described. Validation of the accuracy of the technique and specific clinical applications are presented. It is concluded that this technique provides a rapid means of evaluating left ventricular regional wall motion and that the accuracy of the technique is acceptable for clinical application.


A simple method for calculating left ventricular functions from angiographic data using a programmable hand calculator. The end diastolic volume and systolic ejection fraction have gained increasing acceptance as important indicators of ventricular performance. Time consuming calculations and lack of computer facilities have hindered the emergence of these calculations as a routine part of cardiac catheterization studies. The introduction of the programmable hand calculator has provided means for rapid analysis of ventricular volume data in an efficient and inexpensive manner. In this paper the step-by-step procedure for programming the hand calculator is given, as well as instructions for entering raw data and obtaining final calculations. Programs are given for both single plane and biplane cine angiographic studies.


Correlation between structural transformation and cleavage of the major head protein of T4 bacteriophage. We have studied the maturation of T4 polyheads, the aberrant tubular structures related to the capsid of T4 bacteriophage. Conditions have been found under which more than 95% of the major head protein (P23) undergoes the same cleavage that occurs during development of the normal capsid. The concomitant structural changes in the polyheads have been followed using electron microscope image filtering techniques. As a result of the cleavage, a radical transformation of the hexagonal lattice occurs, involving a 10-15% expansion in the lattice dimensions. However, a metastable intermediate state similar to the uncleaved structure has been observed immediately after cleavage of the protein subunits. Some kind of additional physical stimulus seems to be required to trigger the major structural change, which appears to be highly cooperative.


Lack of correlation between agglutinability, the surface distribution of con A and post-confluence inhibition of cell division in ten cell lines. Agglutinability by concanavalin A, distribution of surface-bound concanavalin A, and maximal cell density in monolayer culture were examined under similar conditions in parallel cultures of ten established cell lines. The degree of agglutinability of the cell lines did not correlate with the presence or absence of patching of concanavalin A bound to the cell surface, as determined with a hemocyanin marker. Agglutinability was also not always correlated with the loss of post-confluence inhibition of cell division. Two clones of mouse 3T3 fibroblasts that maintained post-confluence inhibition of cell division and low agglutinability differed substantially with respect to the surface distribution of concanavalin A. Patching of concanavalin A binding sites is neither necessary nor sufficient to explain differences in agglutinability between cell lines.


Methyl labeling of HeLa cell hnRNA: a comparison with mRNA. The majority of the mRNA molecules in HeLa cells contain 1-2 residue(s) of m6Ap and one blocked, methylated 5' terminal "cap" structure. The hnRNA, which is longer than mRNA, contains both m6Ap and caps but 4-6 times as many m6Ap residues per chain. In addition, nuclear molecules contain T2 RNA ase-resistant, methyl-labeled oligonucleotides ("di-" and "tri-" nucleotides) which are not found in mRNA. Some of the dinucleotides may be precursors to the 2'-0-methylated nucleotides in the cap structures. These results are compatible with internal methylation of hnRNA molecules (both m6Ap and 2'-0-methyl) followed by hnRNA cleavage and the addition of the cap structure to generate at least some of the HeLa cell mRNA. It also appears that some hnRNA molecules, which are longer than most mRNA molecules, contain cap structures suggesting the derivation of some mRNA molecules from the 5' regions of hnRNA.


The size of pulse-labeled fibroin messenger RNA. A method has been developed for the isolation of fibroin gene transcripts from total RNA of the silkworm Bombyx mori. It is based on affinity chromatography using Sephadex-bound polynucleotides capable of selectively hybridizing with fibroin mRNA sequences. In vivo pulse labeling of the posterior silk gland for periods of 10-35 min produces labeled heterogeneous nuclear RNA of high molecular weight (greater 40S). Fibroin gene transcripts can be selected from the total hnRNA population by two consecutive passages through the affinity column. Analysis of the column-bound material in denaturing polyacrylamide-agarose gels reveals that the size of pulse-labeled fibroin mRNA is essentially the same (within 5%) as that of mature cytoplasmic mRNA. This holds true for pulses as short as 6 min, where even nascent mRNA can be observed. However, a small shoulder of material is present on the heavy side of the pulse-labeled mRNA, which could be indicative of an extremely short-lived precurosr species. The purified pulse-labeled mRNA (10 min incorporation) has been further analyzed by chromatography in oligo(dT)-cellulose. The data show that the mRNA is polyadenylated within a few minutes after synthesis.


Analysis of the complexity and diversity of mRNA from chicken liver and oviduct. We have analyzed the sequence complexity and diversity of poly(A)-containing mRNA derived from two highly differentiated chicken tissues. Two independent approaches were used in our analyses. The first involves the annealing of cDNA copies of mRNA to a vast excess of the template RNA; the second procedure uses hybridization between highly radioactive single-copy genomic DNA and mRNA. The results obtained using these two experimental approaches are in good accord and reveal the presence of 12,000-15,000 diverse mRNA species in both chicken liver and oviduct. In both cell types, the kinetics of annealing of cDNA to its template mRNA demonstrate discrete frequency classes with most of the different mRNA species present in fewer than 10 copies per cell. 70% of oviduct mRNA, however, consists of about 10 abundant RNA species, which probably are responsible for the synthesis of the egg white proteins. The diversity of mRNA species in chicken liver and oviduct was further studied by heterologous annealing reactions between cDNA or singlecopy genomic DNA and a vast excess of mRNA. These studies demonstrate that 85% of the different mRNA sequences detected are present in both liver and oviduct, and suggest that the vast majority of the information expressed as mRNA is required for the maintenance of cellular functions common to all tissues.


Effect of cell proliferation on levels and diversity of poly(A)-containing mRNA. The relationship between cell proliferation and the amount and diversity of polyribosome-associated poly(A)-containing messenger RNA [poly(A)+mRNA]has been investigated using a cloned AKR-mouse embryo cell culture system. The following results were obtained. First, an early response to the stimulation of proliferation of AKR-2B cells in culture is a rapid increase in the rate of accumulation of polyribosomal poly(A)+ mRNA. This results in a large increase in the total poly(A)+ mRNA content of rapidly proliferating cells compared to that found in resting cells. Second, the total amount of unique DNA sequence contributing to the poly(A)+ mRNA populations of both growing and resting cells is not detectably different. This corresponds to 9000-11,000 diverse gene equivalents of DNA and represents the transcription of 0.8-0.9% of the haploid mouse genome. Third, most of the increased poly(A)+ mRNA content of growing cells (greater than 90%) reflects an increased rate of production of polysomal mRNA species which are also found in resting cells. Fourth, growing cells appear to contain some species of poly(A)+ mRNA which are either absent or present in very low concentrations in non-growing cells. Within the limits of detection, however, all species of poly(A)+ mRNA present in non-growing cells are also present in growing cells.


Chromatin structure visualization by immunoelectron microscopy. Antibodies elicited in rabbits by chromatin and by purified histone H2B have been used to study the structure of chromatin by immunoelectron microscopy. Chromatin spread on grids reveals a structure of closely packed spherical particles with an average diameter of 104 A, arranged either in clusters or in linear arrays of beads, some of which have a supercoil-like arrangement. No DNA strings connecting the beads could be observed. Upon antibody binding, the diameter of the particles increases up to 300 A. This size is compatible with a model where one layer of gamma globulin molecules 110 A long encircles a sphere of chromatin 100 A in diameter. The presence of rabbit gamma globulins on the enlarged beads has been verified by the addition of ferritin-labeled goat anti-rabbit gamma globulins. Anti-chromatin sera which react with nonhistone proteins but not with free histones or DNA react with more than 95% of the beads; this suggests that most of the beads contain nonhistone proteins. Since the number of nonhistone proteins is large, it is improbable that each sphere contains a full complement of these proteins. We therefore suggest that the various chromatin spheres contain different types of nonhistone proteins. About 90% of the chromatin spheres reacted with antibodies to histone H2B, suggesting the most of the chromatin beads contain this type of histone.


Quantitation of turnover and export to the cytoplasm of hnRNA transcribed in the Balbiani rings. Some quantitative parameters of the intranuclear metabolism and export to the cytoplasm of Balbiani ring 1 and 2 RNA molecules in salivary gland cells of Chironomus tentans have been determined. Growing RNA chains in the Balbiani rings attain uniform labeling with RNA precursors after 20 min of incorporation. The specific activity of 75S RNA released from the Balbiani rings into the nuclear sap increases rapidly and reaches a maximum level between 90 and 180 min of labeling. After 20 min, labeled 75S RNA enters the cytoplasm and accumulates at a linear rate. However, only a small proportion of the RNA produced at the Balbiani ring loci can subsequently be recovered in the nuclear sap (14-17%) or cytoplasm (4-7%) as 75S RNA; presumably the remainder is degraded entirely. Experiments using inhibitors of elongation (actinomycin D) or initiation (5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole) revealed that no significant quanity of the 75S RNA transcribed can be chased into the cytoplasm. Both the kinetics of entry of labeled 75S RNA into the cytoplasm-that is, a constant rate of increase after a brief lag-and chase data are incompatible with a precursor-product relationship between the great majority of nuclear 75S RNA and cytoplasmic 75S RNA with messenger characteristics. The results are discussed in relation to the possibility that a post-transcriptional control mechanism is operating in these cells.


Appearance and disappearance of acetycholine receptor during differentiation of chick skeletal muscle in vitro. During differentiation of embryonic chick skeletal muscle in culture, elaboration of acetylcholine receptor (AChR) and acetylcholinesterase occurs shortly after myoblast fusion. During further development, AChR was found to decrease markedly on the myotube surface, while acetylcholinesterase continued to increase. Surface distribution of AChR, as followed by autoradiography using 125I-alpha-bungarotoxin, was homogeneous in newly fused myotubes. With further differentiation, clusters of AChR appeared on the surface of the myotubes, and their subsequent disappearance paralleled a decrease in overall AChR levels. Quantitative autoradiography showed a reduction of over 75% in the density of AChR on the surface of well differentiated, cross-striated myotubes. Thus the appearance of AChR on the cell surface, its condensation into clusters, and finally its depletion seem to be sequential events in the differentiation of skeletal muscle in culture in the absence of direct neuronal influence.


Low molecular weight viral RNAs transcribed by RNA polymerase III during adenovirus 2 infection. Nuclei isolated from human cells productively infected with adenovirus 2 have been shown to synthesize four low molecular weight RNA species which hybridize efficiently to viral DNA. One species corresponds to the 5.5S or VA RNA (Ohe, Weissman, and Cooke, 1969), and is designated V156. The other three species are novel and have been designated V200, V140, V130, since they are approximately 200, 140, and 130 nucleotides in length, respectively. These viral RNAs retain their distinct electrophoretic properties after denaturation with formamide. RNA species with electrophoretic mobilities similar to those of the V200, V156, and V140 RNAs have been found in the cytoplasmic fraction of cells at late times after adenovirus infection. In isolated nuclei, the V200, V156, V140, and V130 RNAs are all synthesized by DNA-dependent RNA polymerase III, since synthesis is sensitive to high but not to low concentrations of alpha-amanitin. The synthesis of these low molecular weight RNAs continues for a prolonged period of time in isolated nuclei, suggesting that reinitiation occurs. Adenovirus 2 DNA fragments obtained by digestion with restriction endonucleases Eco RI and Sma I were used to map the location of the DNA sequences which encode the RNAs. All the low molecular weight RNAs hybridized to a region of the genome between o.18 and 0.38 fractional lengths from the left end of the adenovirus genome, suggesting that the respective DNA sequences are clustered. Other nonviral low molecular weight RNAs are synthesized in nuclei isolated from infected cells. These include the cellular 5S rRNA species which was minitored by its hybridization to purified 5S DNA from Xenopus laevis.


A new species of virus-coded low molecular weight RNA from cells infected with adenovirus type 2. A virus-coded low molecular weight RNA (5.2S), which migrates slightly faster on polyacrylamide gels than the well characterized adenovirus-specific 5.5S RNA, has been isolated from cells infected with adenovirus type 2. Hybridization-competition experiments and RNA fingerprints indicate that the two virus-associated (VA) RNAs differ in their primary structures. The gene for 5.2S RNA is located to the right of the gene for 5.5S RNA, on the I strand of a DNA segment which extends between positions 30.3 and 32.2 on the map of adenovirus type 2 DNA. Both 5.5S and 5.2S RNA can be detected early after infection and also in the presence of cytosine-arabinoside or cycloheximide. After the onset of viral DNA replication, the synthesis of 5.2S RNA levels off, whereas 5.5S RNA is synthesized in increasing amounts. Both 5.2S and 5.5S RNAs are synthesized in isolated nuclei by an enzyme which resembles RNA polymerase III in its sensitivity to alpha-amanitin. In isolated nuclei, both RNA species are labeled with beta-32P-labeled GTP, which suggests that they are initiated at separate promotor sites.


A direct estimate of the number of human gamma-globin genes. The number of genes specifying human gamma-globin has been determined directly by hybridization of complementary DNA to total human DNA. The complementary DNA was enriched in sequences specific for gamma-globin genes by transcribing globin mRNA isolated from fetal reticulocytes with viral reverse transcriptase, and collecting the material which does not back-hybridize to adult globin mRNA. When hybridized in cDNA excess to DNA, very similar values are found for gamma-gene number as for beta-gene number, suggesting two or at most three gamma-globin genes per haploid human genome. This indicates that the non-Mendelian ratios of gamma-chain mutants found in heterozygotes are due to transcriptional or post-transcriptional regulation rather than to gene dosage. The number of each major human globin gene has now been determined directly by molecular methods.


Small RNA species of the HeLa cell: metabolism and subcellular localization. The small molecular weight RNAs of the HeLa cell have been located in specific subcellular fractions. SnA is located in the nucleolus and is partially bonded to nucleolar 28S RNA. SnD, the most abundant of the small nuclear RNAs, is partially released from the nucleus when the nuclear preparation is briefly warmed. SnF is released from the nuclei when chromatin is digested with the micrococcal nuclease and not when pancreatic DNAase is used. The remainder of the small nuclear species remain in the nucleus following the digestion of chromatin and are concluded to be elements of the "nuclear skeleton." SnK is found predominantly in the cytoplasm, but migrates quantitatively to the nuclear fraction in the presence of high levels of actinomycin D. ScL is totally cytoplasmic and is partially bound to cell membranes. It is the 7S RNA found in oncornavirus virions. All the small nuclear RNAs appear initially in the cytoplasmic fraction before fixation in the nucleus. Two short-lived cytoplasmic species behave kinetically as precursors to the stable nuclear RNAs.


The effect of heat shock on RNA synthesis in Drosophila tissues. Total RNA from Drosophila imaginal discs, labeled under conditions of heat shock, is analyzed by hybridization in situ to salivary gland polytene chromosomes. Grain densities over the hybridizing bands are compared, showing that the response to heat shock is similar for several disc types and fly stocks. Alteration of the culture medium used for labeling during heat shock results in the specific induction (in discs, salvary glands, or fat body) of one of the heat-shock loci to a level far beyond that normally seen. We discuss the implications of this specific induction regarding the mechanism of response. We also discuss the observed difference between transcription labeling of salivary gland chromosomes and labeling by in situ hybridization.


The translational capacity of deadenylated ovalbumin messenger RNA. We present evidence that the poly(A) sequence at the 3' end of ovalbumin mRNA has an effect on its translational efficiency in a reticulocyte lysate cell-free system. Polynucleotide phosphorylase has been used to remove selectively the poly(A) while leaving the rest of the molecule intact. It is shown that the stability of the mRNA in a cell free system is not appreciably affected by this procedure. Measurements of the size of ovalbumin-synthesizing polysomes, rate of peptide elongation, and number of rounds of translation per messenger show a generally reduced efficiency for deadenylated mRNA compared to native mRNA. No comparable difference was observed in experiments with a wheat germ cell-free system, which gives few rounds of translation per mRNA. This indicates that the effect results from a lowering of the efficiency of reinitiation on deadenylated mRNA.


The RNA polymerase II of an alpha-amanitin-resistant Chinese hamster ovary cell line. Amal, an alpha-amanitin-resistant mutant of the Chinese hamster ovary cell line, contains an RNA polymerase activity which elutes from DEAE-Sephadex at a salt concentration characteristic of an RNA polymerase II, but which is not sensitive to alpha-amanitin at levels where the polymerase II of wild-type cells is strongly inhibited. This result suggests that Amal owes its amanitin-resistant phenotype to a mutation affecting one of its genes for RNA polymerase II. To test this hypothesis, we purified the enzyme from Amal and then compared its properties with those of the wild-type enzyme. The mutant enzyme is indeed a polymerase II, and is over 600 times less sensitive to alpha-amanitin and more thermolabile than the wild-type enzyme.


Biochemical characterization of azetidine carboxylic acid-resistant Chinese hamster cells. We have isolated variants of Chinese hamster lung (CHL) fibroblasts with decreased sensitivity to the toxic proline analog, L-azetidine-2-carboxylic acid (AZCA). Resistance to AZCA is a stable characteristic of these cell lines since it is maintained after 40 generations in nonselective medium. Two AZCA-resistant cell lines overproduce and excrete more proline than wild-type cells. Furthermore, AZCA rapidly inhibits the synthesis of proline from glutamic acid in wild-type but not the resistant cell lines. These studies suggest that resistance to AZCA in these cell lines is due to an alteration in some component of the proline biosynthetic pathway, possibly a regulatory site that is inhibited by AZCA.


Insulin receptors in normal and transformed fibroblasts: relationship to growth and transformation. The insulin receptors in normal and transformed lines of mouse Balb/3t3 fibroblasts have been studied. In the normal fibroblasts, the binding of insulin was low in growing cells and increased 2-9 fold in confluent stationary cells. Insulin binding was increased whether growth arrest was due to contact inhibition of growth or serum starvation. When serum-starved cells were stimulated to grow by the addition of fresh serum, insulin binding declined. In cells transformed by simian virus 40, Kirsten, Moloney, and Harvey sarcoma viruses, methylcholanthrene, X rays, or spontaneously, the binding was low, in the same range as growing normal cells. In simian virus 40-transformed cells, insulin binding increased 4 fold as the cells reached higher densities in culture. No relationship to changes in cell size was found. The differences in binding were due to changes in the concentration of the receptors, without changes in their affinity for the hormone.


Kinetics of formation of 5' terminal caps in mRNA. A kinetic analysis of the labeling of the methylated components of messenger RNA and heterogeneous nuclear RNA in mouse L cells indicates that the 5' terminal cap I structures (m7GpppXmpYp) of mRNA are derived from 5' terminal cap structures of hnRNA. Most of the hnRNA caps are conserved during processing, whereas only a portion of the internal m6A residues in hnRNA are conserved. The  cap II structures (m7GpppXmpYmpZp), which constitute the 5' termini of some mRNAs, arise by a "secondary" methylation that occurs after the mRNAs have entered the cytoplasm. This secondary methylation is apparently restricted to a particular subclass of mRNAs having a high frequency of pyrimidine nucleotides at position Y, a composition at position X which differs from that of the bulk of the cap I-terminated mRNAs, and a relatively slow rate of turnover.


A reinvestigation of 5' leads to 3' polarity in 40S ribosomal RNA precursor of Xenopus laevis. The 5' leads to 3' polarity of the 40S precursor rRNA molecule relative to the location of the 18S and 28S RNA regions in the precursor has been reinvestigated. Fragments of rDNA derived by the restriction endonuclease EcoRI and cloned in E. coli were partially digested with the exonuclease induced by bacteriophage  lambda and with exonuclease III from E. coli. The resulting rDNA fragments with single-stranded tails were hybridized separately with 18S and 28S rRNA, and the formation of the hybrid was monitored by determination of radioactivity and by electron microscopy. Since the location of the EcoRI sites in rDNA is known, and the specificity of the two exonucleases for 5' and 3' ends of DNA strands has been established, the hybridization of the different partially digested rDNA fragments with either 18S or 28S rRNA could be interpreted in terms of polarity of the coding strand of rDNA, and consequently of the RNA (see models in Figure 1). The results supports the following model for the rRNA precursor moleculre: 5' end-transcribed space-18S gene-transcribed space-28S gene-3' end.