Changes in lactate dehydrogenase enzyme pattern in Chinese hamster cells infected and transformed with Simian virus 40.

The lactate dehydrogenase (LDH) isozyme patterns of consecutive passages of Chinese hamster embryo cultures were monitored. At early passages the population displayed two LDH bands, M4 and M3H; however, at higher passages the cultures exhibited M2H2, M2H, and M4. When primary cultures of Chinese hamster embryo cells were infected with simian virus 40 (SV40), no change in the LDH pattern was observed; however, the total activity of LDH increased. Twenty-three of 25 transformed colonies isolated from SV40-infected primary cells by their ability to grow in methyl cellulose produced only M4 or M4-M3H isozymes bands. Four of the SV40-transformed clones that produced only the M4 isozyme were tested for LDH activity and found to have activities 2.5 to 3 times greater than the control cells. Chinese hamster kidney epithelial cells transformed with SV40 virus had a decrease in the H subunit production, from 57 to 31%, compared with normal kidney epithelial cells. This decrease in H subunit production led to an increase in the cathode-migrating isozymes. Therefore, a shift to the cathode-migrating isozyme was observed in SV40-transformed cells. This change in LDH pattern might represent a reversion to the enzyme pattern present in fetal cells.


Isolation of variant cells from SV-40 transformed human diploid fibroblasts.

Variant cell lines that are sensitive to density-dependent inhibition of growth have been isolated from three of four SV40-transformed human fibroblast cell lines. Variants were isolated by plating the transformed cells at low density, by treating them with 5-fluorodeoxyuridine, or by growing them on glutaraldehyde-fixed monolayers of normal cells. The variant cell lines, isolated at a frequency of about 2% of all cells forming colonies after treatment, were initially recognized by colonial morphology, and the variant pheno-types were confirmed, after subculturing, by saturation-density determinations. The variant cell lines reach saturation densities that are 40% or less than those of the parent cell lines, and plate in soft agar medium at reduced efficiency, compared with the parent cells. They retain SV40 T antigen. The modal chromosome numbers of two of the variant cell lines were increased, compared with those of the parent cell lines; two other variants were indistinguishable in chromosome number from the parent cells. Stability of these properties over a 6-month period was demonstrated with two of the variants.


5-(3,3-Dimethyl-1-triazeno)imidazole-4-carboxamide (NSC-45388) in the treatment of solid tumors in children.

5-(3,3-Dimethyl-1-triazeno)imidazole-4-carboxamide (NSC-45388) was administered to 46 children with various solid tumors which were resistant to conventional therapy. Two or more courses of NSC-45388 were administered to 13 of 18 children with neuroblastoma, seven of 11 children with rhabdomyosarcoma, three of four children with Wilms' tumor, one of three children with Ewing's tumor, and six of ten children with miscellaneous neoplastic disorders. Major toxic effects included nausea, vomiting, decreased hemoglobin level, thrombocytopenia, and leukopenia. A therapeutic regimen of 200-450 mg/m2/day for 5 consecutive days can be administered safely every 22 days. Objective responses were observed in three children with neuroblastoma and in one child with rhabdomyosarcoma. This drug has minimal but definite activity as a single agent in children with advanced neuroblastoma and rhabdomyosarcoma and should be evaluated further in combination therapy.


Carcinogenicity of nitrosation products of ephedrine, sarcosine, folic acid, and creatinine.

Carcinogenic activity of several synthetic N-nitroso compounds was evaluated in C57BL/6J X C3HeB/FeJ F1 mice. Test substances, suspended in trioctanoin, were injected i.p. in three equal doses given on Days 1, 4, and 7 after birth and animals were held without further treatment for up to 85 weeks. Nitrosoephedrine at a total dose of 600 mg/kg induced metastasizing liver cell carcinomas in 28 of 30 animals. Nitrososarcosine (225 mg/kg) induced similar tumors in 8 of 14 animals. Nitrosofolic acid (375 mg/kg) induced lung adenocarcinomas in 4 of 28 mice. Creatinine-5-oxime (600 mg/kg) showed no evidence of carcinogenic activity. Diethylnitrosamine (12 mg/kg given in four doses), included as a positive control, caused metastasizing liver cell tumors in 23 of 25 animals.


Chemotherapeutic management of bronchogenic carcinoma: trials with NSC-140117, a new methanesulfonic acid ester of aminoglycol.

A new drug, NSC-140117, was used in the management of 15 patients who had late bronchogenic carcinoma and were unable to receive surgical or radiotherapeutic treatment. Good responses in the form of 90% shrinkage in the size of the lesionswere encountered in nine patients (60%). Response is still being maintained from 6 months to 1.5 years after the start of therapy. No toxic effects, including hematopoietic toxicity, were encountered.


Ultrastructural characteristics of human neurilemoma cell nuclei.

Nuclei of human neurilemoma cells exhibit deep and extensive invaginations of part of their surface. Such invaginations contain cytoplasmic matter. However, in areas of the nucleoplasm distant from the invaginations, small membrane-bound bodies, some of which contain a "nucleoid," occur either singly or grouped together and enclosed within a large membrane body. These small bodies are not considered virus-like. Degenerated nuclei from cultured tumor tissue contain spherical bodies, 130 to 230 nm in diameter, with spikes on their surface similar to those seen on envelopes of herpes-type viruses. Significance of these bodies in vivo and in vitro tumor tissues is not known.


Pancreatic islet-cell and other tumors in rats given heliotrine, a monoester pyrrolizidine alkaloid, and nicotinamide.

Three rats of six males, surviving 22 to 27.5 months after one or two intragastric doses of the monoester pyrrolizidine alkaloid, heliotrine (230 mg/kg body weight), and pretreatment with nicotinamide (350 mg/kg body weight) by pretreatment with nicotinamide (350 mg/kg body weight) by i.p. injections, developed pancreatic islet-cell tumors, accompanied in one of the rats by transitory cell papillomas of the urinary bladder and interstitial testicular tumors and in another by a hepatoma. The lesions in the livers showed progression from megalocytosis, to microscopic hepatocellular hyperplasia, to increasingly larger nodules and hepatoma. One rat, given heliotrine, but no nicotinamide, also developed adenoma of the pancreatic islet cells. Adenomas of the pituitary were present among the experimental and also among the control rats killed between 19 and 27.5 months after the beginning of the experiment, and they are not likely to have been caused by the alkaloid. Heliotrine, in which the crucial double bond in the pyrrolizidine moiety is sterically hindered, appears to be less readily sequestered by the liver and also to affect other organs. Alkylation of nicotinamide at the N-1 position prevents its being reused for coenzyme biosynthesis. Hence, pretreatment of rats with large doses of nicotinamide prevents the depletion of nicotinamide adenine dinucleotide coenzymes and liver necrosis in rats given heliotrine (230 mg/kg body weight).


Estradiol-dependent collagenolytic enzyme activity in long-term organ culture of human breast cancer.

An organ culture method suitable for the maintenance of viable human breast cancer for at least 14 days has been described. This method was applied to a total of 94 breast cancer specimens. It allowed good survival of "soft" tumors of various histological types, with loose connective stroma even in hormone-free medium. In contrast, "scirrhous" cancers showed poor survival in hormone-free medium; viable cells were maintained only at the very periphery of the explants. Supplementation of the medium with insulin (10 mug/ml), ovine prolactin (5 mug/ml), and hydrocortisone (1 mug/ml) in various combinations seemed to induce enlargement of viable cancer cells and moderate loosening of the stroma in some cases. However, it did not improve the survival of central tumor cords in scirrhous explants. Further supplementation of the medium with 17 beta-estradiol (minimum effective dose, 0.1 to 10 ng/ml), although it did not affect soft tumors, markedly improved survival of the cancer cells of scirrhous tumors throughout the whole explants, with evidence of collagen digestion around the neoplastic cells. This was observed in 18 of 20 scirrhous cancers subjected to this treatment. Estradiol need not be present during the whole culture period; the results at 14 days were identical in explants treated with estradiol for the first 7 days only or for the entire period. Addition of purified collagenase during the first 24 or 48 hr of culture resulted in complete dissolution of the collage. After such treatment, culture under the usual conditions resulted in excellent survival of the explants without improvement from hormone supplementation; thus, while estradiol was necessary when collagen was present, it was not longer required after collagen digestion. It can be concluded that breast cancer cells in organ culture are only slightly, or not at all, hormone dependent for survival, provided that they are not restrained by a dense collagen barrier. The estrogen-induced changes allowing survival inside the scirrhous explants strongly suggest the presence of an estrogen-dependent collagenolytic enzyme system in the collagen-rich breast cancers. This system could represent an important component of the hormone dependency of human breast cancer growth.


A comparative study of inner membrane enzymes and transport systems in mitochondria from R3230AC mammary tumor and normal rat mammary gland.

Mitochondria from a rat mammary tumor (R3230AC) have been compared with mitochondria from pregnant and lactating rat mammary glands, with particular attention paid to inner membrane enzymes and Transport proteins. In the tumor the mitochondrial adenosine triphosphatase was not activated by 2,4-dinitrophenol, in contrast to the mammary mitochondria from lactating or pregnant rats. Translocation of adenosine diphosphate across the inner membrane was found to be more rapid in the tumor by virtue of lovered Km adenosine diphosphate and raised Vmax. Transport of phosphate and dicarboxylic acids occurred at similar rates in all three types of mitochondria. The inner membrane proteins were also examined directly by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and some differences are noted. These results, although they indicate subtle differences between the inner mitochondrial membranes of tumor as compared with those of pregnant or lactating rat mammary glands, cannot form the basis of an explanation for enhanced glucose utilization and aerobic lactic acid production in this tumor.


Turnover of high-molecular-weight cell surface proteins during growth and expression of malignant transformation.

The turnover of cell surface proteins in normal rat kidney cells transformed by a temperature-sensitive Rous sarcoma virus has been studied by polyacrylamide gel electrophoresis and autoradiography using cell monolayers prelabeled by lactoperoxidase-catalyzed radioiodination. Labeling of serum-starved cells under conditions that are nonpermissive for the expression of transformation reveals most of the radioactivity in the 250,000 molecular weight region. Parallel labeling of cells simultaneously exposed to serum limitation, under conditions that are permissive for the expression of transformation, reveals some radioactivity in the same slow-migrating region, but most of the label appears in the two faster migrating regions. The relative turnover of such external proteins has been investigated by examining the relative alterations in iodinated proteins after addition of normal levels of serum to a medium of serum-starved cells. There is a greater relative turnover of the high-molecular-weight external component under conditions in which ther transformation phenotype is expressed, as compared with conditions that limit the expression of transformation.


Clonal isolation of epithelial cells from mouse lung adenoma.

Clones of epithelial-like cells were established from urethan-induced mouse lung adenoma. Electron microscopy of one clone showed that the cells contained lamellar inclusion bodies similar in appearance to those seen in the adenoma precursor, the type II alveolar pneumocyte. The clones exhibited characteristics associated with both "transformed" and "normal" cells in culture; i.e., although aneuploid, the cells grew at a slower rate than most transformed cells, did not form colonies in soft agar and, after prolonged subculture, were not tumorigenic when transplanted s.c. into appropriate hosts. Hydrocortisone treatment of the cloned cells led to growth stimulation and the eventual acquisition of neoplastic potential. Epithelial tumors were produced more readily in athymic, nude mice than in antilymphocyte serum-treated A/He mice. The cells are producing a C-type RNA virus into the culture medium.


Rosetting of antibody-sensitized tumor cells with anti-immunoglobulin antibody-coated erythrocytes by a new method for detecting antigens on cells.

Tumor cells were treated with rabbit antibody to tumor-associated cell surface antigens and tested with erythrocytes coated with antibody specific for the sensitizing rabbit immunoglobulin. The sensitized tumor cells formed rosettes with the indicator cells. By this method, we confirmed that line 1 and line 10 hepatoma cells (from two tumors independently induced by diethylnitrosamine in strain 2 guinea pigs) bear antigens not present on normal liver cells. We also confirmed that line 1 and line 10 cells bear antigenically different tumor-associated cell surface antigens. This method appears simpler than other serological methods for detecting tumor-associated cell surface antigens on tumor cells. Also, this method may be a general one for detecting and enumerating cells bearing surface antigens.


Morphological patterns of primary nonendocrine human pancreas carcinoma.

The study of histological sections of 406 cases of nonendocrine pancreas carcinoma at Memorial Hospital indicated that morphological patterns of pancreas carcinoma could be delineated as follows: duct cell adenocarcinoma (76%), giant-cell carcinoma (5%), microadenocarcinoma (4%), adenosquamous cancinoma (4%), mucinous adenocarcinoma (2%), anaplastic carcinoma (2%), cystadenocarcinoma (1%), acinar cell carcinoma (1%), carcinoma in childhood (under 1%), unclassified (7%). In 195 cases of patients with pancreas carcinoma, search was made for changes in the pancreas duct epithelium and these were compared to duct epithelium in a control group of 100 pancreases from autopsies of patients with nonpancreatic cancer. The following incidences were found for pancreas cancer and nonpancreatic cancer, respectively: mucous cell hypertrophy, 39 versus 28%; pyloric gland metaplasia, 28 and 17%; epidermoid metaplasia, 6 and 12%; papillary hyperplasia, 42 and 12%; atypical duct hyperplasia, 14% and none; cancinoma in situ in 19% and none in the control group. Mucin in the majority of pancreas cancers suggested that the cell type of origin of the common pancreas cancer is the mucin-producing duct epithelium. The association of atypias and carcinomas in situ in the patients with pancreas carcinoma implies, by analogy to other organs, that there may be a significant latent period between the appearance of carcinoma in situ and the grossly recognizable pancreas cancer.


Acetoacetate coenzyme A transferase activity in rat hepatomas.

The presence of succinyl-coenzyme A:acetoacetate CoA transferase (CoA transferase) (EC 2.8.3.5), an initiator of ketone body utilization in nonhepatic tissue, was examined in liver from normal, partly hepatectomized, neonatal, and tumor-bearing rats, as well as in a series of transplantable rat hepatomas ranging widely in growth rate. While levels of CoA transferase are extremely low in normal, host, and regenerating liver, considerable amounts of activity are detectable in neonatal liver and in the hepatomas. In fact, the content of CoA transferase in the series of Morris hepatomas increases progressively with increase in tumor-growth rate. The fastest-growing tumor studied (7288Ctc) contains about the same amount of CoA transferase activity as rat skeletal muscle (i.e., an activity of about 0.1 mumole of acetoacetate used per min per g tissue). These results clearly indicate that the faster-growing hepatomas have adequate capacity to utilize ketone bodies in bioenergetic or biosynthetic activities. Furthermore, the enzymes from normal and hepatoma 7288Ctc tissues are quite similar with respect to (a) size of about 10(5) daltons, (b) reaction mechanism requiring formation of an enzyme:CoA intermediate (from ping-pong kinetic data), and (c) various kinetic parameters (such as Michaelis constants, product competitive inhibition constants, and acetoacetate substrate inhibition). The enzymes from rat skeletal muscle and Morris hepatoma 7288Ctc have the same isoelectric point (7.6), which differs from that for the rat heart enzyme (6.8).


Tumor-selective inhibition of the incorporation of 3H-labeled amino acids into protein by cyanate.

Sodium cyanate at a dose level of 125 or 250 mg/kg i.p. caused an inhibition of incorporation of 3H-labeled amino acids into cytoplasmic and nuclear proteins of the rapidly growing hepatoma 7777 and the slowly growing hepatoma 9618A. There was no inhibitory effect on 3H-labeled amino acid incorporation into protein in the livers of rats bearing these tumors. Studies on the effects of sodium cyanate on incorporation of 3H-labeled amino acids into total acid-insoluble material indicated that a greater than 85% inhibition could be achieved in hepatoma 5123C, hepatoma 9618A2, and the MK3 kidney tumor with either little or no effect in host liver, kidneys, brain, skeletal muscle, intestinal mucosa, and regenerating liver after partial hepatectomy.


Comparison of the cytidine 5'-diphosphate and adenosine 5'-diphosphate reductase activities of mammalian ribonucleotide reductase.

A comparison of the cytidine 5'-diphosphate (CDP) and adenosine 5'-diphosphate (ADP) reductase activities from Ehrlich tumor cells was made to determine if the properties of the enzyme for these substrates were the same, except for the allosteric effector. It was observed that various purification steps did not result in an enzyme fraction that had a constant ratio of CDP:ADP reductase activities. The optimal Mg2+ ion concentration for CDP reduction was 3 to 4 mM, while the optimal Mg2+ ion concentration for ADP reduction was 0.1 mM inhibited ADP reduction. CDP reduction was relatively insensitive to the presence of dimethylformamide or dimethyl sulfoxide in the reaction mixture, but ADP reduction was decreased in the presence of these two compounds. Periodate-oxidized adenosine 5'-monophosphate, on incubation with the enzyme, had a greater effect on CDP reduction but little or no effect on ADP reduction. The response of the CDP and ADP reductase activities to the same negative effector was essentially the same. Both CDP and ADP reductions showed similar decreases in the presence of various concentrations of deoxyadenosine 5'-triphosphate. These data suggest that the Ehrlich tumor cell reductase enzyme system could consist of at least two different enzymes that may be regulated by the same allosteric protein.


Rat mammary gland atypia produced by iodine blockade with perchlorate.

Prior published work from our laboratory concluded that there was a need for appropriate metabolic activity of iodine in breast tissue for normal growth and development. Results from studies in rats that were made iodine deficient showed histological changes in the breasts that were atypical and dysplastic. These tissue findings were further affected by the presence of estrogen and thyroxine. These changes parallel the iodine uptake of the tissues, thus representing a difference in the utilization of iodine by the mammary glands. Using an ion blockade agent, sodium perchlorate, breast tissues lacking iodine were evaluated by both endocrine and histological techniques. A dose-response series was completed that showed that perchlorate therapy for 8 weeks at 400 mg/100 ml produced breast blockade by a reduction in iodine uptake of greater than 52% of the control. At these levels, the histological experimentation showed atypia and some pleomorphism of the cells, particularly in the glands of the lobules. Blockade was less effective in estrogen-treated groups. It is especially notable that both histological changes and uptake reduction were greatest in those breasts that had been rendered euthyroid by thyroxine replacement, thus clearly indicating the necessity of iodine itself for maintenance of normal breast development. By this blockade the responses of iodine inadequacy in the breast were shown to cause abnormal tissue changes relative to the percentage of the block obtained.


Preferential inhibition by homopolyribonucleotides of the methylation of ribosomal ribonucleic acid and disruption of the production of ribosomes in a rat tumor.

The literature indicates that some mechanism other than the interferon or host-mediated immune enhancement might also be responsible for an antitumor effect of polyinosinate-polycytidylate [poly(I)-poly(C)]. We have examined the effect of this drug on the synthesis of ribosomes and other macromolecules in a rat tumor, the Novikoff ascites hepatoma. The nucleolus was one of the primary targets affected by the administration of poly(I)-poly(C) in vivo. A progressive decline of the activity of nucleolar ribosomal RNA methylases began within 2 hr, followed by a decline of the nucleolar RNA content. The activity of nucleolar RNA polymerase was inhibited only at later time intervals. Labeling of tumor macromolecules in vivo revealed that the methylation of ribosomal RNA and the production of ribosomes, particularly in the small subunits, were immediately and progressively affected, followed by inhibition of the synthesis of DNA, RNA, and protein at later times. In addition, poly(I)-poly(C) also induced disaggregation of polyribosomes and restricted the movements of nuclear RNA to cytoplasm and of cytoplasmic protein to nucleus. These in vivo effects of poly(I)-poly(C) on tumor cells was observed neither on the host livers nor on livers of normal rats. Studies on isolated nucleoli showed that the in vitro addition of polyinosinate and several other compounds actively inhibited tumor ribosomal RNA methylases but were devoid of inhibitory effect against liver ribosomal RNA methylases; these results augment other studies in the literature in suggesting a selective effect of the polyinosinate moiety on tumor cells. We conclude from this study that initial impairment of the methylation of ribosomal precursor RNA, following exposure of tumor cells to poly(I)-poly(C), is responsible for the destruction of ribosomes, preferentially the small subunits, during the maturation processes. Failure to provide new ribosomes thus triggers the events limiting the growth of tumor cells.


Hormone dependence and estradiol receptors in the D series of mammary nodule outgrowth lines and tumors.

Preneoplastic mammary nodule outgrowth lines were examined for their ability to grow and produce tumors in ovariectomized BALB/c mice. In addition, these nodule outgrowth lines and tumors derived from them were investigated for cytoplasmic estrogen receptor proteins by qualitative and quantitative techniques. Early ovariectomy, performed within 4 weeks after transplantation, slightly delayed but did not permanently block the ability of three different nodule lines to completely fill the fat pad with nodule tissue. Ovariectomy performed later than 4 weeks after transplantation or adrenalectomy performed early or late had no effect on nodule growth. Neither early nor late ovariectomy or adrenalectomy had an effect on the maintenance of the nodule alveolar phenotype. Ovariectomy performed 3 weeks after transplantation had little effect on the tumor potential of the low oncogenic line D1 or the high oncogenic line D2. Samples of nodule outgrowths transplanted from ovariectomized mice responded to a chemical carcinogen in a similar manner, as did nodule outgrowths transplanted from control experiments. Thus, the altered hormonal environmenment in ovariectomized mice apparently did not select for subpopulations of nodule cells with altered tumorigenic potentials. The ovarian independence of the nodule lines and tumors derived from them was correlated with a very low level of cytoplasmic estrogen receptor. These results, along with other data, illustrate the nature of the independence on ovarian hormonal control for tumorigenesis in these mammary tumor virus-free nodule lines.


5'-nucleotide phosphodiesterase isoenzyme in patients with hepatitis B infection.

The hypothesis that hepatitis B infection is etiologically related to hepatoma has been investigated by studying the interrelationships between hepatitis B surface antigen (HBsAg, Australia antigen) and the fast-moving 5'-nucleotide phosphodiesterase Band V isoenzyme (5'-NPDase-V). Sera from 58 patients with viral hepatitis were tested for 5'-NPDase-V and HBsAg. The isoenzyme was found in 34 of 37 patients who were also positive for HBsAg but in only 4 of 21 hepatitis patients who were HBsAg negative. Five patients convalescing from hepatitis were negative for both HBsAg and the isoenzyme. Preparative gel electrophoresis showed that these 2 markers were different proteins. Of 34 hepatoma patients, 29 were positive for 5'-NPDase-V. Only 1 isoenzyme-positive patient was positive for HBsAg by counterimmunoelectrophoresis. However, of 16 isoenzyme-positive hepatoma patients available for radioimmunoassay, 8 were NBsAg positive (50%). None of 21 hepatoma samples tested for antibody to NBsAg was positive. Of 21 "normal" carriers of HBsAg and 10 carriers with Down's syndrome, 4 persons were detected with the isoenzyme. The results suggest that HBsAg and 5'-NPDase-V in the presence of liver damage are associated and thus provide a new marker enzyme between hepatitis B infection and hepatoma.


Properties of acidic saccharides produced by B16 melanoma cells treated with 1-methyl-3-isobutylxanthine.

A cyclic nucleotide phosphodiesterase inhibitor, 1-methyl-3-isobutylxanthine, promoted the differentiation and maturation of B16 melanoma cells, phenomena associated with biological alterations in the surface properties of the cells. 1-Methyl-3-isobutylxanthien inhibited cell replication and increased the intracellular content of melanin and cyclic adenosine 3':5'-monophosphate. Significantly greater amounts of sialoglycoproteins were associated with 1-methyl-3-isobutylxanthine-treated cells. However, the total amount of [3H] glucosamine incorporated into anionic polysaccharide (both sialoglycopeptide and mucopolysaccharides) was not significantly changed.


Comparison of physical and immunological properties of plasma membranes of two mouse leukemia cell lines, P388 and L1210.

A method for the isolation of the plasma membranes of the mouse leukemic cell P388 is described. The method includes surface labeling of the cell membrane with 125I. The 125I specific activity of the plasma membrane preparation is 10- to 13-fold higher than that of the cell homogenate; enzyme assays and electron microscopy further corroborate the purity of the P388 plasma membrane preparation. The predominant iodinated membrane proteins have molecular weights of 85,000, 66,000, and 13,000 daltons. Antibodies prepared against P388 and L1210 plasma membranes inhibit the growth of the corresponding cells in the absence of complement. They show cross-reactivity to each other but do not affect the growth of HeLa cells.


Changes in lymphoid cyclic adenosine 3':5'-monophosphate metabolism during murine leukemogenesis.

Cyclic adenosine 3':5'-monophosphate (cyclic AMP) levels were slightly increased in preleukemic AKR mouse thymus cells, compared with nonleukemic thymus cells, but were markedly reduced in leukemic cells. Adenylate cyclase activity rose during the preleukemic and leukemic phases of leukemogenesis. Although the drop of epinephrine-induced stimulation of thymus adenylate cyclase in the preleukemic phase was probably age related, there was an additional decrease of adenylate cyclase activation by epinephrine in leukemic cells. Cyclic AMP phosphodiesterase activity was slightly higher in preleukemic cells and more than fourfold AKR thymus. These observations suggest that cyclic AMP phosphodiesterase is largely responsible for the low levels of cyclic AMP in leukemic cells. Significant changes in cyclic AMP metabolism are already detectable before neoplastic cells may be found in the thymus.


Sarcoma-negative leukemia-positive transformed cell culture established from a murine sarcoma virus-induced rat bone tumor.

Inoculation of the Soehner-Dmochowski isolate of the Moloney strain of murine sarcoma virus (MSV), designated MSV-SD, consistently leads to the development of bone tumors in the susceptible New Zealand black (NB) rats. Two separate cell cultures have been established from 2 individual MSV-SD-induced NB rat bone tumors. Cells of 1 bone tumor culture, designated RBT-E, are in early in vitro passages. These cells form colonies in agar medium and take up 2-deoxy-D-[3H]glucose at a greatly enhanced rate, 5 times that of normal nontransformed rat embryo cells. Cells of the RBT-E culture release both MSV and murine leukemia virus (MuLV) and therefore contain sarcoma-positive leukemia-positive transformed cells. The other rat bone tumor culture, designated RBT-L, produced MSV at early passages. RBT-L culture has been passaged over 130 times in vitro. Cells of the RBT-L culture form colonies in agar medium and take up 2-deoxy-D-[3H]glucose at an enhanced rate (3 times that of rat embryo cells), indicating the presence of transformed cells within the RBT-L culture. However, cells of the RBT-L culture at late passages (Passage 130 or more) produce only MuLV and no detectable MSV activity (as shown by the lack of tumor-inducing activity and the lack of focus-forming activities by direct assay or by infectious center assay). Attempts to rescue MSV activity from RBT-L cells by cocultivation with MuLV-producing mouse cells were not successful. The MuLV found in the RBT-L cells, however, is a competent helper virus capable of rescuing the MSV genome from MSV-SD-induced hamster bone tumor cells. All the available evidence supports the notion that late passages of the RBT-L culture contain transformed cells that do not produce conventionally detectable MSV. These cells are referred to as sarcoma-negative leukemia-positive cells. The sarcoma-negative leukemia-positive cells represent a different kind of MSV-induced transformed cells and provide a unique system for studies in search of MSV markers such as MSV-specific antigens and MSV-specific nucleotide sequences.


Morphological differentiation of cultured mouse glioblastoma cells induced by dibutyryl cyclic adenosine monophosphate.

A culture line of mouse glioblastoma cells changed morphologically to differentiated astrocyte-like cells when cultured in medium with dibutyryl cyclic adenosine monophosphate and theophylline. Morphological alteration occurred within only 5 hr when 3 mM dibutyryl cyclic adenosine monophosphate and 1 mM theophylline were used, and in 1 day when 1 mM theophylline were used. Cells showing this morphological change reverted completely to immature cells when they were transferred to medium without these two chemicals. Addition of 1 or 3 mM dibutyryl cyclic guanosine monophosphate with 1 mM theophylline to the medium also induced development of cytoplasmic processes from these cells and the cells became stellate, although the cytoplasmic processes were not as long or as numerous as those induced by dibutyryl cyclic adenosine monophosphate, and the altered cells could not be referred to as differentiated glia cells. Sodium butyrate induced morphological alterations similar to those induced by dibutyryl cyclic guanosine monophosphate, but fewer cells showed these alterations. Addition of cyclic adenosine monophosphate or cyclic guanosine monophosphate in the presence of theophylline or addition of theophylline alone did not induce morphological changes of the cells.


Cyclic adenosine 3':5'-monophosphate levels and activities of related enzymes in normal and leukemic lymphocytes.

The role of cyclic adenosine 3':5'-monophosphate (cyclic 3':5'-AMP) in the regulation of cell division in lymphocytes obtained from healthy donors and from patients with chronic lymphocytic leukemia (CLL) was investigated by determining the levels of cyclic 3':5'-AMP and glycogen and also the activities of several enzymes that are closely associated with the metabolism of these cellular components. Intracellular levels of cyclic 3':5'-AMP were measured in normal and CLL lymphocytes in nondividing, dividing, and quiescent [after phytohemagglutinin (PHA) addition]states. In normal lymphocytes the levels of cyclic 3':5'-AMP fluctuated throughout the cell cycle after PHA addition, whereas in CLL lymphocytes the levels were approximately 3-fold lower than in normal cells and remained relatively constant before, during, and after mitogenic stimulation. Normal cells contained approximately 3-fold lower levels of glycogen than CLL cells, whereas glycogen phosphorylase activities were increased 2- to 4-fold above those in nondividing cells in normal but not in CLL lymphocytes after stimulation with PHA. Furthermore, cyclic 3':5'-AMP phosphodiesterase activities were higher in CLL lymphocytes than in normal ones. Collectively, these studies demonstrated that (a) the intracellular levels of cyclic 3':5'-AMP differ in these two cell types; (b) the levels of cyclic 3':5'-AMP and glycogen qualitatively correlate with the activities of the enzymes that are related to these components; and (c) an inverse relationship between the levels of cyclic 3':5'-AMP and cell growth exists in mitogen-stimulated lymphocytes from healthy donors but not from patients with CLL. These biochemical differences are presumed to exist between normal and "leukemic" lymphocytes, but alternatively they may reflect normal populations of immunologically distinct lymphocytes.


Production of a serine-protease with macrophage migration-inhibitory factor activity by virus-transformed cells and human tumor cell lines.

BALB/c mouse 3T3 cells transformed by simian virus 40 (SV3T3), baby hamster kidney cells transformed by polyoma virus or Rous sarcoma virus, and a range of neoplastic human cell lines release material that inhibits the migration of macrophages and lymphocytes. Similar migration-inhibitory factor (MIF) activity was not detected in supernatants from cultures of untransformed 3T3 or baby hamster kidney cells and a variety of human diploid cell strains. Physico-chemical characterization of the MIF produced by SV3T3 and HeLa cells revealed substantial similarities with the MIF produced by mitogen-activated human peripheral lymphocytes. MIF released by tumor cells is inhibited by pancreatic and soybean trypsin inhibitors and by diisopropylfluorophosphate, indicating that it is a serine-protease. Comparison of MIF produced by SV3T3 cells with a serine-protease plasminogen activator released by the same cells indicated that the latter is more heat labile and has a more heterogenous elution profile after chromatography on Sephadex G-75. The possible role of MIF in causing proteolytic modification of the surface properties of tumor cells and in altering cell-mediated immune responses to neoplastic cells is discussed.


The distribution and properties of aspartyl transfer RNA in human and animal tumors.

The elution profile of aspartyl transfer RNA (aspartyl-tRNA) from reversed phase 5 chromatography for tRNA from a spectrum of animal tissues and tumors and human tumors has been examined. It was found that SV40-induced hamster tumors, BHK21/cl 13 cells in culture, certain carcinogen-induced tumors in the Ehrlich ascites tumor, and a number of human carcinomas and adenocarcinomas contained a distinct increase (3- to 20-fold) in the percentage of a late-eluting aspartyl-tRNA over that found in nonmalignant tissues, other animal tumors, and in human melanomas and sarcomas. The ability of the late-eluting aspartyl-tRNAAspIV to bind to ribosomes in the presence of the codons for aspartic acid was compared to that of aspartyl-tRNAAspIII and was found to be approximately the same. Also, the ability of each of the 4 isoaccepting species of aspartyl-tRNA to engage in ribosomal incorporation of aspartic acid into a polypeptide was determined. All 4 isoacceptors function equally well in the amino acid incorporation.


Human neuroblastoma in nude mice.

For establishment of a reproducible model of human neuroblastoma, 2 to 5 million of established neuroblastoma cell lines (SK-N-SH, SK-N-MC) were injected s.c. or i.p. into 20 nu/nu mice of a predominantly Swiss back-ground. Following latency periods of 8 to 21 days, tumors developed at the injection site and grew to 4-ml volumes within 3 weeks. Histologically, the tumors resembled the original metastases from which the tumors were derived; however, the SK-N-SH appeared to have evidence of morphological differentiation. When compared to monolayer culture, the heterotransplanted SK-N-SH tumor had decreased dopamine-beta-hydroxylase activity and elevated cyclic adenosine 3':5'-monophosphate phosphodiesterase activity. Activity of cyclic adenosine 3':5'-monophosphate phosphodiesterase in the transplanted SK-N-MC tumor was not appreciably different from the activity in the cultured cells. Serum dopamine-beta-hydroxylase levels in the mice bearing SK-N-SH tumor increased threefold. The SK-N-MC cultured cells lacked dopamine-beta-hydroxylase and did not alter existing serum levels in the SK-N-MC tumor-bearing mice. 67Ga injected i.v. was found to localize in the tumor after 24 hr. Human neuroblastoma in the nude mouse can be a reproducible and informative model for tumor pharmacology, screening, radionuclides, tumor localization and imaging, and investigating morphological differentiation.


Evaluation of the cell-mediated immune response to murine sarcoma virus by (125I)iododeoxyuridine assay and comparison with chromium 51 and microcytotoxicity assays.

The cell-mediated immune response of C57BL/6 mice to murine sarcoma virus (MSV) was examined by the [125I]-iododeoxyuridine release cytotoxicity assay using MSV-induced sarcoma tissue culture cell lines as target cells. Cellular cytotoxicity was detected as early as 3 days after virus inoculation. Most mice assayed between 12 and 17 days after MSV inoculation gave positive results with maximum levels of activity present on Days 13 and 14. Reactivity was frequently detected for up to 100 days after MSV inoculation, although at low levels (5 to 10%). Additional experiments comparing the kinetics of the cellular response as measured by different in vitro cytotoxicity assays were performed. The results showed a good direct correlation between the [125I]iododeoxyuridine release assay and a 51Cr release assay. A similar pattern of reactivity was also observed when the cellular response was measured by a visual microcytotoxicity assay, although reactivity dropped off more rapidly and became undetectable in most instances by 20 days after injection of MSV. Studies on effector cell type revealed that cytotoxicity in all three assays was T-cell dependent, being eliminated by treatment with anti-theta plus complement. Macrophages did not appear to play a role, since treatment with carbonyl iron and magnet had no effect.


Organized inclusions in astrocytic and amorphous inclusions in neuronal mitochondria of human frontal brain tissue.

Brain tissue specimens were obtained early post mortem from the frontal lobe of seven unselected elderly subjects known to have been free of neurologic disease. Electron microscopically, two types of intramitochondrial inclusions were seen in four of seven cases. In two cases a few astrocyte mitochondria of the gyral white matter showed dense, elongated inclusions with an ordered linear substructure. These inclusions were, as a rule, accompanied by a row of prismatic cristae. In three cases some nerve cell mitochondria contained amorphous material of medium density and compact appearance. The globular masses often occupied the whole width of the mitochondrion. A relationship between the observed finding and a particular disease or morbid condition was not apparent. The inclusions are regarded as the morphologic substrate of a nonspecific metabolic change or degenerative process of the mitochondrion.


Annulate lamellae and crystalline inclusions in granular endoplasmic reticulum of the islet organ and associated tissues of a cyclostome, Myxine glutinosa.

Cytoplasmic annylate lamellae were found in the islet organ of a cyclostome, the hagfish (Myxine glutinosa), predominantly in cells interpreted as young proliferating beta-cells, and also in endocrine cells and enterocytes of the bile duct and gut and in the endothelial cells of small blood vessels. A close association was observed annulate lamellae and granular endoplasmic reticulum. Both in cells with and in those without annulate lamellae, crystalline inclusions of proteinaceous nature were seen in granular endoplasmic reticulum. These inclusions were occasionally closely associated to annulate lamellae, and a direct continuity could be seen between granular endoplasmic reticulum and the outer nuclear membrane surrounding an inclusion partially situated in the perinuclear cisterna. Rod-shaped structures and rounded electron dense bodies were seen in the nuclei of some islet parenchymal cells. The presence of annulate lamellae in the islet organ and associated tissues of Myxine glutinosa is believed to be related to the very high phylogenetic age of this species. The close association observed between annulate lamellae, granular endoplasmic reticulum, crystalline inclusions, and sometimes also nuclear membranes, may be of functional significance.


Characterization by different techniques of adrenocorticotropin and gonadotropin producing cells in lerot pituitary (Eliomys quercinus).

Adrenocorticotropin and gonadotropin producing cells were localized in the adenohypothysis of normal Lerots by using anti-beta 1-24 ACTH, anti-LH, anti-LH beta, anti-PMSG antisera.  In order to study their fine structure two techniques were employed: a superimposition technique which consists of detailed comparisons between the same cells in light, fluorescence and electron microscopic preparations and an immunocytochemical technique on ultra-thin sections using the peroxidase anti-peroxidase complex technique.  The superimposistion technique allows an excellent description of cell ultrastructure of individually identified cells of each type.  With this method we were able to describe the corticotropin secreting cells as lucent cells with electron dense granules ranging in size from 2500 to 3500 A.  The gonodotropin secreting cells are darker and their granules are about 2000 A in diameter.


Light- and electron-microscopic studies on the secretory cytology of the adenohypophysis of the Japanese quail, Coturnix coturnix japonica.

The effects of thyroidectomy, adrenalectomy, and castration on the pars distalis of male Japanese quail, and of injection of LH-RH on sexually inactive females, were investigated by light and electron microscopy. Correlation between light and electron microscopy was attained by use of alternate thin and thick sections. Six types of secretory cells were identified and the ultrastructural characteristics described. Putative endocrine functions have been designated on the basis of responses to experimental interventions and on other criteria. The putative STH cells are characterized by the presence of large dense secretory granules (250-300 nm) that are stained with orange-G by the trichrome method. They occur only in the caudal lobe and appear to be unchanged by castration, thyroidectomy, adrenalectomy and LH-RH injection. The putative prolactin cells are characterized by large (400-600 nm), spherical or polmorphic, dense secretory granules stainable with acid fuchsin and aniline blue; prominent Golgi apparatus and well developed endoplasmic reticulum with densely packed, regularly parallel lamellae. They are found mainly in the cephalic lobe. The prolactin cells develop some vacuolization after adrenalectomy and undergo some degeneration after castration. The ACTH cells, which are restricted to the cephalic lobe, are identified by the dense, spherical granules (250-300 nm) that are stained with acid fuchsin. After adrenalectomy, they lose their secretory granules and are transformed into large, chromophobic adrenalectomy cells. TSH cells are so designated by their response to thyroidectomy including loss of their fine secretory granules and transformation to large, vacuolated thyroidectomy cells. We have found TSH cells and thyroidectomy cells only in the cephalic lobe. Basophilic cells, considered to be gonadotropes, occur in both the cephalic and caudal lobes. The gonadotropes of the cephalic lobe appear to have slightly larger (120-200 nm) granules than the caudal lobe (120-150 nm). However, after castration, the gonadotropes in both lobes become hypertrophied and vacuolated and are transformed into mutually indistinguishable castration cells. Twenty minutes after injection with LH-RH, the gonadotropes of both lobes increase in size and number, degranulate, develop vacuoles in the cytoplasm, and appear very similar to castration cells.


Cell types in the adenohypophysis of the Japanese quail and effects of injection of luteinizing hormone-releasing hormone.

The cells of the adenohypophysis of the Japanese quail were studied by both light and electron microscopy after exposure to long photoperiods or injection of lutenizing hormone-releasing hormone (LRH). Six cell types were identified in the adenohypophysis by examining alternate thick and thin sections by light and electron microscopy. In the cephalic lobe, there are four types of glandular cells. They are the prolactin cells, ACTH cells, TSH cells, and gonadotropic cells (FSH?). In the caudal lobe, there are two types of cells, STH cells and gonadotropic cells (LH?). After exposure to long daily photoperiods, gonadotropic cells in both lobes were strongly activated. They became larger and accumulated many granules. ACTH cells became vacuolated; granules were sparse. Synthetic LRH injection (10 mug/0.2 ml/day) for 10 days to the non-photostimulated quail stimulated certain numbers of the gonadotropic cells in the both lobes, although the response of the cells was less than that induced by photostimulation. No response was seen in the other cell types.


Vacuolated pinealocytes in the hedgehog (Erinaceus europaeus L.) and the mole (Talpa europaea L.).

Special cells in the hedgehog and mole pineal gland which are characterized by an enormous single vacuole were studied. The presence of secretory granules (in the hedgehog) and of paracrystalline structures (in the mole) in the cytoplasm of these cells indicates that they are pinealocytes.


Ultrastructural study of the neural fat-body system in the cockroach Periplaneta americana.

The neural fat-body system of the ventral nerve cord in the cockroach Periplaneta americana was studied with the light and electron microscopes. This adipose tissue surrounds the connectives and extends over the ganglia. The adipose cells typically contain numerous extremely large lipid inclusions, pleomorphic lysosomes, and tightly packed glycogen granules. The neural lamella consists of a thick inner layer rich in collagen fibers and a thin outer layer of granular material. At points where the fat body is attenuated, this granular layer is split and the outer lamina is reflected superficially to ensheath and apparently to anchor the fat body.


The ultrastructure of topical cutaneous calcinosis.

Mineralized plaques, which develop at the site of repeated subcutaneous injections of 100 mug KMnO4/0.2 H2O in rats, were investigated by electron microscopy. The newly formed, delineated, white plaque tissue at the injection site consisted of numerous, mostly unaltered fibroblasts and collagen fibers, without participation of inflammatory cells. Some signs of cell injury were found in the center of the lesions. Numerous, irregularly distributed, small, mineralized foci were seen near the fibroblasts. These were formed by aggregation of small needle-like units (50 A in diameter and 0.05-2.0 mum long). These needle-shaped units were found either in vesicular, cell derived structures, considered to be shed cell fragments, or on collagen fibers. Intramitochondrial deposits of such needle like units were seen frequently. Fusion of smaller mineralized foci to larger plaques occured and then needle-shaped units were seen at the periphery of the electron-dense lesions. Hypotheses concerning the mechanism of experimental cutaneous calcinosis (soft tissue mineralization) are discussed and related to the findings of this study. Probable intracellular crystal deposition and mineralization in cell-derived structures were shown for the first time in topical cutaneous calcinosis.


Histochemical, ultrastructural and hormonal studies on the pars distalis of the echidna (Tachyglossus aculeatus).

There were no consistent significant differences between the concentrations of luteinizing hormone (LH) and adrenocorticotrophin (ACTH) in the rostral compared with the caudal zone of the echidna pars distalis. This suggests that LH is secreted by cells containing S-type granules (probably corresponding to secretory vesicles 200-300 nm diameter) which are distributed throughout the gland. Some of the cells containing vesicles 100-200 nm diameter, seen in small numbers in both zones of the gland, may be responsible for the secretion of ACTH. The concentration of pituitary LH is in the range of that found in eutherian mammals, but the concentration of ACTH is lower than that reported for other vertebrates, and this may be linked causally with the remarkably low rate of corticosteroid secretion in the echidna. The absence of significantly increased levels of pituitary LH and ACTH in a chronically orchidectomized and adrenalectomized animal adds to other evidence which suggests that mechanisms involving a negative feedback of steroid hormones on the hypothalamo-hypophysial axis may not be fully developed in the echidna.


Ultrastructure of the budgerigar testis during a photoperiodically induced cycle.

Spermatogenesis in the Budgerigar can be arrested by reducing the birds' photoperiod to 8 hours of daylight or less. When this occurs, Sertoli cell cytoplasm shows a great increase in the size and number of residual bodies, while the smooth endoplasmic reticulum is reduced. If the bird is kept at 8 hours of daylight for some weeks large lipid droplets are seen in Sertoli cytoplasm, and degenerated spermatids are apparently phagocytosed. The interstitium shows fewer active Leydig cells, a paucity of lipids and occasional ovoid mitochondria. The basal lamellae of the tubule which are thick and convoluted before and during spermatogenesis become thinner and straighter. It is thought that these morphological changes reflect changes in metabolic activity.


On degeneration of peptidergic neurosecretory fibres in the albino rat.

Three types of degenerating peptidergic neurosecretory fibres have been found in the posterior pituitary of chronically dehydrated albino rats. "Dark" neurosecretory fibres and their swellings contain neurosecretory granules, neurotubules, shrunken mitochondria and diffusely distributed fine dense material. Some swellings are filled with synaptic vesicles and/or conglomerations of dense membranes. The transitional forms exist between these fibres and extracellular accumulations of electron dense material. Synaptic vesicles, single neurosecretory granules, lipid-like droplets and lamellar bodies occur in the latter. Some neurosecretory fibres and swellings have numerous polymorphous inclusions arising due to degradation of secretory inclusions and organelles, mitochondria and neurotubules in particular. "Dark" neurosecretory elements and those with numerous polymorphous inclusions are enveloped by pituicyte cytoplasm. Sometimes the plasma membranes both of the pituicytes and neurosecretory fibres are destroyed or transformed into a multi-membrane complex. It is assumed that pituicytes may phagocytize degenerating neurosecretory elements. N  urosecretory fibres with a locally dissolved neuroplasm and/or large lucent vacuoles seem to be due to axonal degeneration by the "light" type. These neurosecretory elements, the largest of them in particular, may transform into large cavities bordered by a membrane and containing flake-like material and single-membrane vacuoles. Degeneration of neurosecretory elements seems to occur mainly due to hyperfunction of the hypothalamo-hypophysial neurosecretory system.


Adrenocortical response in the duck exposed to corticosteroid administration and salt loading.

The functional zonation in the adrenocortical tissue of the duck was experimentally investigated after chronic administration of corticosteroids of different types (corticosterone, desoxycorticosterone, dexamethasone) and salt loading (chlorides of sodium and potassium). The cytomorphology of the interrenal cells belonging to subcapsular and central zones was explored by light- and electron microscopy and by biochemical analysis of plasma corticosterone. Corticoid-induced involution of the interrenal tissue, or hyperactivity elicited by salt loading were evident in both subcapsular and central regions of the gland. In the duck the adrenocortical tissue appears to be devoid of steroidogenic functional differentiation. The modifications of different cellular organelles and inclusions can be explained in the light of known concepts about corticosteroid metabolism. The depression of plasma corticosterone titre in corticosteroid-treated animals agrees with the image of cytological inhibition. The lowered corticosterone value in KCl-loaded ducks signifies increased peripheral metabolism of this hormone. The hypercorticosteronemia in NaCl-loaded ducks is probably related to activation of the nasal gland under osmotic stress.


Ultrastructural studies on the intranuclear inclusions of the rabbit adenohypophysis in vivo and in vitro.

Intranuclear inclusions of three basic types are described in various parts of the rabbit adenohypophysis in vivo and in organ and cell cultures. These inclusions are mostly found in the postnatal tissue but occur in both glandular and non-glandular cells. A comparison is made with similar structures encountered elsewhere. Their significance is not known.


Different secretory responses of periinsular and other acini in the rat pancreas after pilocarpine injection.

Histological, histochemical, and electron microscopical investigations of the rat pancreas, one to five hours after pilocarpine injection (4 mg in 1 ml normal saline solution), showed different secretory responses in periinsular and apoinsular acinar cells. The responsiveness to parasympathetic stimulation is markedly retarded and weakened in the periinsular area. It is supposed that the halo phenomenon is related not only to an inhibition of the secretion, but also to a longer lasting synthesis of zymogen granules. The insular hormones, glucagon and insulin, which are possibly locally enriched by insulo-acinar capillary anastomoses or by diffusion, may be involved in the regulation of activity in the periinsular cells.


Inhibition of protease activity in cultures of rous sarcoma virus-transformed cells: effect on the transformed phenotype.

We have examined the role of proteolytic activity in the genesis and maintenance of the transformed phenotype by growing cultures of chick embryo fibroblasts transfromed by Rous sarcoma virus either in medium containing plasminogen-free serum or in medium to which protease inhibitors were added. Alterations in morphology, adhesiveness, and hexose transport were used as markers for the transformed state. Addition of the trypsin inhibitors NPGB or Soy Bean Trypsin Inhibitor at concentrations which inhibited transformation-associated fibrinolysis restored adhesiveness and morphology to near normal, but did not affect the rate of hexose transport. Growth of Rous-infected cells in plasminogen-free medium blocked the appearance of morphological and adhesive alterations, but allowed the rate of hexose transport to increase to the transformed level. Thus we were able to separate the appearance of transformation-specific changes in morphology and adhesiveness (which apparently require fibrinolytic activity) from the increased rate of hexose transport (which is independent of fibrinolytic activity). Another trypsin inhibitor, TLCK, although it did not inhibit fibrinolysis, was very effective at restoring adhesiveness and morphology as well as hexose transport to normal. This raises the possibility that there is another, perhaps earlier, protease involved in the genesis of the transformed phenotype.


Nucleic acid hybridization using DNA covalently coupled to cellulose.

We describe a method for linking RNA and DNA covalently to finely divided cellulose through a diazotized aryl amine, which reacts primarily with guanine and uracil (thymine) residues of single strands. The high efficiency of coupling and high capacity of the cellulose for nucleic acid make possible a product with as much as 67 mug of nucleic acid per mg of cellulose. The product is especially suitable for hybridization experiments where very low backgrounds are important, and it is stable in 99% formamide at 80 degrees C so that hybridized nucleic acid can be recovered easily. Full length linear Simian Virus 40 (SV40) DNA, produced by cleavage of SV40(I) DNA with S1 nuclease, can be coupled to diazo cellulose with an efficiency of 80-90%, and is effective in hybridization experiments with SV40 DNA, complementary RNA synthesized in vitro from SV40(I) DNA with E. coli RNA polymerase, and the SV40-specific fraction of total RNA from SV40-infected and transformed cells. In these experiments an excess of cellulose-bound DNA was used, and the efficiency of hybridization was about 90% when ribonuclease treatment of the hybrids was omitted.


Effects of cycloheximide on virus RNA replication in an inducible line of polyoma-transformed rat cells.

In this article, we describe two distinct effects of cycloheximide (CH), a potent inhibitior of protein synthesis, on the replication of polyoma virus (PV) DNA in an inducible line of PV-transformed rat cells (LPT cells). Exposure of LPT cells to CH causes up to an 8 fold increase in the cellular concentration of PV DNA determined by molecular hybridization. The same treatment inhibits cell division and chromosomal DNA replication. However, the amount of chromosomal DNA per cell is not affected by the drug. In LPT cells treated with mitomycin C (MMC), PV DNA replication is enhanced after 7 hr. During the period extending from 7 hr to 24 hr, the concentration of virus DNA increases at least 100 fold. CH added to the cells 0-7 hr after treatment with MMC inhibits the replication of PV DNA by 90-100%. The inhibition is less effective in cells exposed to CH from 7 hr and on. The inhibitory effect is reversible: virus DNA synthesis is resumed after removal of CH from the growth medium. Thus CH acts as an inducer of virus DNA synthesis in cells whose resident viral genome is repressed, but inhibits the autonomous replication of the activated genome following induction with MMC.


Fibrin overlay methods for the detection of single transformed cells and colonies of transformed cells.

Fibrin overlay methods are described which can detect the plasminogen activator produced by single transformed cells or small colonies of transformed cells. These methods were applied to malignant cells derived from humans, mice, hamsters, rats, and chicks. The lysis observed was plasminogen dependent. Transformation of chicken cells by Rous sarcoma virus was detected 4 days after infection. The number of lysis zones produced was proportional to the virus inoculum and was identical to the number of morphologically determined foci. These methods may also have application in model systems for scoring transformation by chemicals. Transformed mouse and chicken cells were detected at the single cell level and the number of lysis zones produced was dependent on the number of cells present, the time of incubation, and the concentration of plasminogen.


Determination of limb bud chondrocytes during a transient block of the cell cycle.

The process of determination is studied by using a cell culture system derived from dissociated chick embryo limb buds. When limb bud cells obtained from embryos younger than stage 25 (undertermined) are temporarily prevented from passing through the cell cycle (either by maintaining the cells on a petri dish or in the presence of high concentrations of cyclic AMP, both of which depress thymidine-H(3) incorporation), some cells subsequently form cartilage colonies. These results support the hypothesis that a temporary block at some stage in the cell cycle causes mesoblasts to acquire the capacity to differentiate into cartilage cells.


Adaptation of the mitochondrial systems of Rhodotorula gracilis to low oxygen pressure.

The obligate aerobic yeast, Rhodotorula gracilis, was grown in a liquid minimal medium at 1 mm Hg partial pressure of oxygen, conditions under which growth (measured as the increase in total protein) is reduced to 30% of the maximum rate. A significant increase in the ratio between mitochondrial oxidative enzymes and total protein occurs rapidly under these conditions. A concurrent increase in the ratio area of mitochondria/area of cytoplasm was also observed. The relative increase in mitochondrial enzymes, oxidase activities and mitochondrial membranes is due to the inhibition affecting the increase in cytoplasmic structures more significantly than the increase in mitochondrial structures at low pO2. The difference between mitochondrial and cytoplasmic syntheses cannot be ascribed to changes in the availability of ATP but it might rest with some other oxygen-utilising process (pyrimidine redox coenzymes, synthesis of sterols). The experimental conditions studied appear to offer a valuable tool for the investigation of the relationships between mitochondrial and cytoplasmic structures.


The inhibitory effect of glucose on the differentiation of trophic Hartmannella culbertsoni into viable cysts.

During encystation of Hartmannella culbertsoni induced by taurine or epinephrine, 60-70% of the reserve glycogen is degraded. Glycogen phosphorylase is activated and glycogen synthetase is inhibited after 6-8 hr of exposure to the encystation medium. The carbon skeleton of glycogen but not that of protein is utilised in the synthesis of cyst wall cellulose. Exogenously added glucose (225 and 550 mM) blocks encystation, degradation of glycogen and synthesis of cellulose. Cyclic AMP synthesis is also very much reduced in cells exposed to glucose.


[Small-cell carcinoma of the larynx].

In a 69-year-old man by biopsy and for the first time on necropsy a primary small-cell carcinoma of the laryngeal ventricle with metastases into the liver was detected and confirmed. Under the electron microscope in the cells endocrine granules were found which were not argyrophil.


Reversibility of lysosomal and glucose 6-phosphatase changes produced in the rat liver by dimethylnitrosamine.

The present investigation was undertaken to discover whether repeated doses of dimethylnitrosamine (DMNA) could produce a cumulative toxic effect on the rat liver. For this purpose doses were selected at a level just too low to produce cytopathological changes, as indicated by depression of glucose-6-phosphatase and induction of autophagic vacuoles (AV) in hepatocytes, when given once only. Single subcutaneous injections of 10 or 3 mg/kg induced these cytopathological changes in the centrilobular (CLB) hepatic cells but when the dose was reduced to 1 mg/kg no such changes were seen. After daily administration of 1 mg/kg for 4 or 8 weeks we observed both glucose-6-phosphatase depression and autophagy, and in addition there was marked hypertrophy of the rough endoplasmic reticulum, nucleolar microsegregation and the appearance of distorted, often ring-shaped mitochondria with shortened cristae. Kupffer cells exhibited a marked increase in lysosomal activity. With the exception of mitochondrial changes and Kupffer cell activity this same picture was observed, although in milder form, when the dose administered was 0.3 or 0.1 mg/kg daily for the same period. When treatment was continued for 12 weeks, however, the only differences from control rats were the presence of hypertrophied rough endoplasmic reticulum (RER) at all three dose levels, nucleolar microsegregation at the upper two dose levels, and pronounced Kupffer cell activity at the top dose. These findings indicate that cumulative cytopathologic effects occur only up to 8 weeks at the dose levels studied but hypertrophy of RER and increased Kupffer cell activity persist up to 12 weeks.


[Enzymatic characteristics of the catalytic subunit of the protein kinase complex of human lymphocytes].

In earlier reports we have shown the existence in human lymphocytes homogenate, of a cyclic-AMP dependent protein-kinase activity. We demonstrate by affinity chromatography that two subunits display respectively cyclic-AMP binding and phosphorylating properties. Divalent cations such as Ca++, Mg++ or Mn++ are required for enzymatic activity. ATP which is an obligatory cosubstrate acts as an inhibitor when its concentration is higher than 10(-6)M.


[The binding of tritiated dexamethasone to lymphocytes of normal and leukemic subjects].

Specific binding sites for 3H-dexamethasone, possessing the characteristics of glucocorticoid receptors, were demonstrated in normal human circulating lymphocytes.  The number of glucocorticoid receptors was found different from normal in half of the acute or chronic lymphoid leukaemias studied.  A correlation between the number of glucocorticoid receptors and the therapeutical benefit of the glucocorticoid administration will be performed, including these preliminary observations.


[Standardization of "exchange" techniques to measure estradiol receptors in extracts exposed to biospecific adsorbants].

The measurement of the binding of steroid hormone receptors to biospecific adsorbants requires the development of "exchange" techniques. Two types of techniques based on the principle of differential dissociation were standardized: solid phase exchange with hydroxylapatite and liquid phase exchange with adsorption of the ligand to charcoal dextran. In both cases, binding of oestradiol to its receptor was measured in calf uterine cytosol extracts. Results obtained by both techniques were comparable. The half-times of the oestradiol-receptor complex dissociation determined from exchange kinetics are on the same order as those obtained by direct methods used in previous studies.


[Radioimmunologic assay of plasma corticotropin in the green frog, Rana esculenta L].

In the green Frog (Rana esculenta) the plasma contains a polypeptide immunologically related to human and porcine corticotropins. A radiommunoassay capable of detecting 4.10-12 g hog ACTH has been used for a direct plasma ACTH assay in the Frog. Using this method, the ACTH rate was determined both in untreated frogs and in animals under various experimental conditions.


[The effect of gonadotropic hormones and the fetal hypophysis on testosterone production by the testis of 18 day mouse fetuses in organ culture].

The production of testosterone (measured by radioimmuno-assay) by the 18-day-old mouse fetal testis may be stimulated specifically by ovine LH (1 ng, p less than 0.005) and HCG in organ culture. A stimulation by FSH is observed only with high doses (10 mug, p less than 0.0005). Prolactin and ACTH have no effect. Age-matched fetal pituitaries increase significantly the testosterone production in the culture medium (p less than 0.0005).


[Plasmids controlling the resistance to several antibiotics in C. perfringens type A, strain 659].

A strain of C. perfringens type A, isolated from a patient, was found to be resistant to four antibiotics: tetracycline (Tet), chloramphénicol (Chl), erythromycin (Ero) and clindamycin (Cli). Clones resistant to only two drugs (Tet-Chl or Ero-Cli), or sensitive to all drugs were found in cultures of the wild-type strain treated by acridine dyes or ethidium bromide. DNA analysis by equilibrium centrifugation confirmed that the original strain contains two resistance plasmids, one called Rip 401 (Tet-Chl) and the other Rip 402 (Ero-Cli), which represent respectively 1.8 +/- 0.2% and 2.3 +/- 0.2% of the total amount of DNA in this strain of Cl. perfrigens.


[The effect of alloxan on the multiplication of different types of normal or cancerous cells, and on the transformation of chick embryo fibroblasts by Rous virus in vitro].

The action of alloxan was studied in vitro, on different categories of normal or cancerous cells. At concentrations of 250 and 350 gamma/ml, alloxan does not significantly inhibit the growth of normal cells, whereas it does inhibit the growth of cancerous cells. Furthermore, alloxan inhibits infection and transformation of chicken-embryo fibroblasts infected by the Rous virus.


Disappearance of bradykinin in the renal circulation of dogs. Effects of kininase inhibition.

In chloralose-anesthetized dogs, we investigated the disappearance of bradykinin on passage across the renal circulation. The peptide was infused into a renal artery at various doses (5-200 ng/kg min-1); renal blood flow and the concentration of kinins in renal venous blood were then determined and the percent survival of bradykinin on passage through the kidney calculated. Bradykinin caused a dose-related increase in renal blood flow, urine flow, sodium excretion, and kinin content of renal venous blood. Intravenous administration of BPP9alpha (300 mug/kg), a peptide kininase II inhibitor, potentiated the renal vasodilator, diuretic, and natriuretic actions of bradykinin and augmented the survival of the kinin on passage through the kidney from 12.72 +/- 1.64% in control dogs to 53.92 +/- 7.48% (P less than 0.001). Furthermore, the values of peptide survival were positively correlated with the increases in renal blood flow (r = 0.92, P less than 0.01), urine flow (r = 0.75, P less than 0.01), and sodium excretion (r = 0.68, P less than 0.01) produced by bradykinin. In addition, BPP9alpha by itself increased renal blood flow (16%, P less than 0.01), urine flow (115%, P less than 0.005), and sodium excretion (167%, P less than 0.02). Similarly, the concentration of kinin in renal venous blood and the excretion of urinary kinins rose from 0.11 +/- 0.03 ng/ml and 4.1 +/- 1.1 ng/min to 0.24 +/- 0.05 ng/ml (P less than 0.005) and 38.5 +/- 12.2 ng/min (P less than 0.02). These studies suggest that kinins generated intrarenally play a role in the regulation of renal blood flow and salt-water excretion and that variations in the capacity of the kidney to inactivate kinins may be a determinant of the intrarenal activity of the kallikrein-kinin system.


Localization patterns of plasma apolipoproteins in human atherosclerotic lesions.

The localization patterns of human plasma lipoproteins and their respective apoproteins and of neutral lipid were determined in normal and atherosclerotic arteries. Specific antisera were prepared against plasma low-density lipoproteins(LDL) and their apoproteins (apoB), high-density lipoproteins(HDL) and one of their major apoproteins (apoA-I1, and apoC-III, which is a major apoprotein of very low-density lipoproteins (VLDL). Using immunofluorescence  techniques, the various antigens were localized in arterial samples obtained at surgery or autopsy. The three apoproteins and neutral lipid were localized to the same tissue areas, namely, lipid core regions and certain connective tissue of atherosclerotic lesions, in 61% of the fibrous plaques and 48% of the fatty streaks examined. In marked contrast, none of the uninvolved arterial regions showed the presence of all four factors together. As controls, the localization of other serum proteins was also determined in these arteries using immunofluorescence techniques. Fibrinogen was associated with regions of maximum complementary localization of factors in 37% of the fibrous plaques and 64% of the fatty streaks. However, albumin was found in only 4-5% of these same regions. The present results suggest that not only LDL but also HDL and VLDL or their respective apoproteins as well as fibrinogen are specifically retained by certain tissue components of the atherosclerotic lesion.


Clinical conference: De subitaneis mortibus. XIII. Multifocal Purkinije cell tumors of the heart.

Multifocal Purkinje cell tumors were found in the heart of a nine-month-old black female infant who died with arrhythmias which had become progressively more frequent and severe until they were completely intractable. The Purkinje cell tumors were composed of exactly the same type of cells found in the left bundle branch and the right bundle branch, and they were also located in the expected region of the His bundle. In none of these locations were these Purkinje cells forming normal longitudinally oriented Purkinje fibers, however, and no such fibers were found anywhere in this heart. The cells of the tumors contained glycogen but not in excess of that normally expected to be present in Purkinje cells. No evidence for a generalized abnormality of glycogen metabolism or storage was present. Except for the Purkinje cells, the remaining myocardial cells of the heart were all normal. The fundamental fault appeared to be failure of the Purkinje cells to organize into the normal histological pattern which is characterized by longitudinally oriented Purkinje fibers. Instead, all the Purkinje cells were rounded or polygonal and generally aggregated together into small discrete nodules of varying size. Future cases of this nature deserve careful attention to the nature of their cardiac rhythm and conduction, and in fatal cases there should be special studies of the histological appearance of their cardiac centers of impulse formation and conduction.


Potential for clinical use of the analytical laser microprobe for element measurement.

Use of the laser microprobe for rapid emission spectroscopic analysis of elements in microscopic samples of biological material is described. The technique depends on vaporization of the microsample with a focused laser beam at a temperature that renders the vapor incandescent for spectrochemical analysis. Spectral line intensities are recorded photographically with densitometry of the negatives or photoelectrically. Current capability permits analysis of about 10(-8) to 10(-10) g of tissue, with detection limits of 10-12 to 10-15 g of element. Groups of elements can be simultaneously analyzed. Minimum sample preparation is required, and the analysis is done on the stage of a light microscope, usually on an air-dried sample on a plastic slide. We exemplify the technique in analysis of gold in cultured fibroblasts treated with gold salts and in human skin after treatment with gold salts for rheumatoid arthritis, in element changes in biopsies of transplanted human hearts, and in unique profiles of groups of elements in human cancer tissue.


Application of electron spin resonance to determination of serum drug concentrations.

Displacement of spin-labeled drugs from drug-specific antibodies in human serum can be measured by use of electron spin resonance. This "spin immunoassay" has advantages of speed, simplicity, and small volume requirements over other standard techniques such as gas-liquid, liquid-liquid, or thin-layer chromatography. Unlike radioimmunoassay, there is no need to separate free and bound label and there are no radiation hazards. We present clinical and experimental data showing how this method can be applied to the routine determination of morphine and diphenylhydantoin concentrations in serum.


Evaluation of serum lipid and lipoprotein levels in normal Chinese. The influence of dietary habit, body weight, exercise and a familial record of coronary heart disease.

The effects of dietary habit, body weight, physical activity and a familial record of coronary heart disease on serum lipid and lipoprotein levels in Chinese were examined. Increased dietary preference for animal meat products was associated with increased serum cholesterol and beta-lipoprotein levels. Increased carbohydrate intake and overweight were associated with increased serum triglyceride and pre-beta-lipoprotein levels. A familial record of coronary heart disease was associated with increased serum total lipid and pre-beta-lipoprotein levels. Based on these results, the factors which influence the serum lipid and lipoprotein concentrations in Chinese are probably similar to those in other ethnic groups.


The effect of heparin on serum lipoprotein-X.

Serum lipoprotein-X was investigated in 12 patients with cholestasis in basal conditions and 20 minutes after intravenous heparin. In all cases, lipoprotein-X was positive in the first sample and became negative after heparin. A decrease in triglycerides was observed in all patients after heparin with an increase in free fatty acids and mobilization of prebetalipoproteins, and a transformation of lecithin into lysoleithin in 4 cases after heparin. These facts suggest that heparin plays an important role in the mobilization of serum lipoprotein-X.


"Sinking" lipoprotein in normal, hyperlipoproteinaemic and atherosclerotic patients.

The presence of a "sinking" lipoprotein (SLP), demonstrated by combining electrophoretic and ultracentrifugal methods, has been recorded in 13.6% of 44 normal subjects, in 62.2% of 37 hyperlipoproteinaemic patients with types IIA, IIB and IV and in 39.8% of 107 atherosclerotic patients. The recorded data suggest that the "sinking" lipoprotein may be related to human atherosclerosis and to hyperipoproteinaemic states. A pre-beta (S band in the fraction with density greater than 1.006 appeared in 2 out of 20 subjects receiving as isocaloric hyperglucidic diet (CHO, 80; F, 5;P, 15) for 8 days and in 3 out of 10 subjects receiving an acute ethanol oral load. The nature of the nutritionally determined lipoprotein is doubtful. It seems improbable that the newly synthetized lipoprotein is the "true" sinking lipoprotein as this considered to be a genetically determined marker. These data may signify that other "sinking" lipoproteins unrelated to LP-Berg system may exist or may be determined. It is also probable that the new synthetized lipoprotein may have some relationship with the "intermediate" lipoprotein.


The biochemical composition and metabolism of lipoproteins in type V hyperlipoproteinaemia.

The biochemical composition of lipoproteins in Type V hyperlipoproteinaemia was investigated with special reference to apolipoprotein-B. The in vivo metabolism of protein-labelled low density lipoprotein was also studied in five such patients (3 diabetic, 2 obese subjects). In association with pathologically elevated plasma levels of chylomicrons and very low density lipoproteins, low density lipoprotein concentration was significantly reduced. The proportions of triglyceride to apo-B and triglyceride to cholesterol were significantly increased in all lipoprotein classes, while the proportion of cholesterol to apo-B was significantly reduced. Disappearance of chylomicronaemia with treatment was associated with a prompt increase in low density lipoprotein concentration. The reduced low density lipoprotein concentration was accompanied by an accelerated fractional turnover rate, and by an absolute turnover rate which was subnormal. These results were interpreted as reduced synthesis of low density lipoprotein apoprotein and as a possible block in the conversion of very low density lipoprotein to low density lipoprotein in some Type V patients.


Isolation of Wilms' tumor antigens by chelation.

A procedure for ethylenediaminetetraacetate extraction of minced Wilm's tumor was assessed as a method for isolating Wilm's tumor antigens. An antigen was detected by immunodiffusion using an adsorbed antiserum to this extract. This antigen was also found in ethylenediaminetetraacetate extracts of in vitro cultures of nephroblastoma cells.


Plasma apolipoprotein A levels in healthy human adults.

The apolipoprotein A concentration in the plasma of ten healthy human subjects was measured immunologically on three occasions at monthly intervals and the following results recorded. 1. There was no difference between fasting and non-fasting apoprotein A concentrations. 2. Apoliportein A levels were higher in females than in males (p less than 0.001). 3. The correlation coefficient calculated for high density lipoprotein cholesterol and apolipoprotein A was 0.62. 4. The apoprotein values varied widely between subjects and ther were considerable concentration differences from month to month in the plasma of each individual.


Serum levels of thyroid-stimulating hormone in hyperlipoproteinemia.

Similar serum lipoprotein abnormalities have been demonstrated in hyperlipoproteinemia type III and hypothyroidism. In this study the levels of thyroid-stimulating hormone in serum were determined in clinically euthyroid patients with different types of hyperlipoproteinemias and in subjects with a normal lipoprotein pattern. The mean value for thyroid-stimulating hormone was significantly higher in the group with hyperlipoproteinemia type III than in normal controls (p smaller than 0.01) and in the group with hyperlipoproteinemia type II A (p smaller than 0.02). The levels of thyroid-stimulating hormone were positively correlated to the triglyceride and cholesterol concentration in the very low density lipoprotein fraction and the ""III-index''.


A dominant syndrome of metacarpal and metatarsal asymmetry with tarsal and carpal fusions, syndactyly, articular dysplasia and platyspondyly.

A mother and daughter with metacarpal and metatarsal asymmetry, tarsal and carpal fusions, long bone articular dysplasia and platyspondyly are reported as a possible autosomal dominant syndrome affecting skeletal development. The asymmetry of the second metacarpals is perhaps the most striking finding and resulted in a range of bone lengths in the two individuals from minus 6.3 to plus 3.7 standard deviations from the mean.


The value of the external carotid circulation in the assessment of intracranial disease.

The external carotid circulation often contributes a vascular supply in intracranial disease. Adequate radiological evaluation of this circulation is important and may alter the surgical management in these patients.


Relation of intracellular K+ and steroidogenesis in isolated adrenal zona glomerulosa and fasciculata cells.

1. Intracellular K+ content, water spaces and corticosterone output were measured in isolated zona glomerulosa and zona fasciculata-reticularis cell suspensions of rat adrenal cortex, after incubation in vitro under conditions designed to alter steroidogenesis. 2. Intracellular K+ of unpurified zona glomerulosa cells was not altered after stimulation of corticosterone output with serotonin. Similarly, with zona glomerulosa cells purified by unit gravity sedimentation, no change in intracellular K+ was detected after stimulation of steroidogenesis with serotonin or angiotensin II. 3. In high-potassium medium (final concentration 8.4 mmol/1), parallel increases in intracellular K+ and corticosterone output were observed with both purified zona glomerulosa cells. However, a similar increase in intracellular K+ also occurred in high-potassium medium with zona fasciculata cells, whose steroid output is unresponsive to external potassium concentration ([K+]). 4. Ouabain at 10(-5) mol/1 depressed the intracellular [K+] of glomerulosa cells but did not alter basal or stimulated corticosterone output. Similar results were obtained with fasciculata cells. 5. Ouabain at 5 times 10(-4) mol/1 further depressed intracellular [K-+] of glomerulosa cells and inhibited basal and stimulated corticosterone output. However, this concentration of ouabain also inhibited steroidogenesis in fasciculata cells. 6. These results demonstrate a variety of situations where changes in intracellular [K+] are dissociated from those in corticosterone output and indicate that intracellular [K+] cannot be the sole mechanism regulating steroidogenesis under these conditions.


Mobilizing in response to a major disaster.

In an attempt to meet the emergency needs of a major disaster in what some have termed "the greatest natural disaster in American history," the National Institute of Mental Health (NIMH) responded immediately by dispatching a mental health team to Wilkes Barre, Pennsylvania, to examine and deal with the mental health needs of flood survivors. After conferring with local mental health workers and rescue officials, the team concluded that there would be long-term psychiatric sequelae of the flood and that NIMH would have a useful and important role in dealing with them. The team made its report to the Director of NIMH, Dr. Bertram S. Brown, who then assigned his executive assistant, K. Patrick Okura, the task of fleshing out the present mental health program of the area and finding out how NIMH resources could best be put to use in meeting the crisis. The NIMH program, in keeping with its tradition, consisted of three parts: service, training, and evaluation. Within a 30-day period a proposal requesting frunds for a year-long program of mental health services to the residents of the flood-affected area was approved and launched. This article describes "Operation Outreach," as the program was called.


Stimulation by angiotensin II of the release of vasopressin from incubated rat neurohypophyses---possible involvement of cyclic AMP.

1. Calcium did not influence the spontaneous release of vasopressin from rat neurohypophyses in vitro when used in concentrations of 0.05, 0.5 and 2.8 mM in the bathing medium. 2. Stimulation of the basal output of vasopressin by angiotensin II (1 X 10(-9) M) required at least 0.5 mM calcium in the medium. 3. Angiotensin II stimulated the release of vasopressin within 2.5 min of incubation, maximal release was observed after 10 min. 4. Angiotensin II rapidly promoted the accumulation of tissue cyclic AMP; maximal accumulation was observed after 5 min of incubation. 5. Theophylline and dibutyryl cyclic AMP produced varying degree of stimulation of the release of vasopressin. 6. Increases in vasopressin secretion and in the accumulation of cyclic AMP were always present when neurohypophyses were exposed to optiman concentrations of angiotensin II. The results presented suggested that cyclic AMP may be an intermediate step for the release of vasopressin by endogenous angiotensin II.


Structural requirements for angiotensin analogues to accumulate cyclic AMP and release vasopressin from the incubated rat neurohypophysis.

1. Angiotensin I, a decapeptide, stimulated the accumulation of cyclic 3',5'-AMP (cyclic AMP) and the release of vasopressin from incubated rat neurohypophyses. 2. Various octapeptides related to angiotensin II were capable of producing similar neurohypophyseal effects. 3. Longer incubation periods were needed with peptides having alterations or omission (e.g. heptapeptide 2-8) at position 1 of the parent molecule to evoke similar effects to those of angiotensin II. 4. Our results suggest strongly that physiological doses of angiotensin-related molecules stimulate the secretion of vasopressin through cyclic AMP, and that the neurohypophyseal receptor responsible for these effects is similar to that involved in their peripheral actions.


Complete Klüver-Bucy syndrome in man.

A 22 year old right-handed man suffered a viral meningoencephalitis, possibly herpetic, resulting in bilateral damage to the temporal lobes as confirmed by appropriate clinical, electrophysiological and neuroradiological studies. Extended clinical neuropsychological evaluation documented all the characteristic features of the syndrome described by Klüver and Bucy following bilateral ablation of the temporal lobes in adult Rhesus monkeys, including "psychic blindness," oral exploration, hypermetamorphic impulse to action," lack of emotional responsiveness, aberrant sexual behavior, and an insatiable appetite. Additionally, a  severe Wernicke's aphasia and a profound memory disorder were evident. The significance of these features as regards limbic function in the human is discussed.


Multinuclide evaluation of hepatic mass lesions.

Radionuclide imaging with labeled colloids is widely used to evaluate and localize primary and metastatic tumors of the liver. The method is fairly sensitive, but the nonspecificity of focal defects remains a significant limitation. Lesions such as cysts and abscesses appear as space occupying areas that are indistinguishable from neoplastic masses. Utilizing a variety of radiopharmaceuticals, one may obtain additional information concerning such lesions. Hepatic blood flow scintiphotography is performed with the Anger camera following the intravenous injection of a high activity, small volume bolus of 99m-Tc pertechnetate. Vascular lesions such as hepatomas or hemangiomas will show increased activity in the lesion which should easily differentiate them from avascular processes such as abscesses, cirrhotic pseudomasses and most metastatic lesions, all of which remain "cold" on these flow studies. If one does not posses a camera, useful blood pool rectilinear scans of these lesions may be obtained with 131-I or 99m-Tc human serum albumin or ionic 113m-In. Additional information concerning the metabolic activity of focal defects on the colloid study is obtained using 75-Se-selenomethionine or 67-Ga. The former is an indicator of active protein metabolism while the latter attaches to lysozymes of metabolically active cells. With either agent, hepatomas show avid uptake, metastatic lesions show variable uptake, and cysts or chronic pseudotumors of cirrhosis show poor uptake. The two agents differ in abscess detection where 75-Se-selenomethionine uptake is poor while 67-Ga concentration generally is intense. 131-I-Rose Bengal occasionally may prove useful in demonstrating impression by an atypically positioned gallbladder or focal dilatation of the biliary tract as a cause of a defect on the colloid scan. Ultrasound examination may complement the radionuclide studies. It is useful for corroborating the presence of lesions and for evaluating their consistency (cystic vs. solid). The information obtained from this multinuclide approach has made scintigraphy examination of the livermore specific. After the completion of this non-invasive series of studies, one generally may venture an intelligent opinion concerning the etiology of the space occupying disease.


Critical review of tomography in radiology and nuclear medicine.

This review covers conventional radiographic tomography, radioisotopic tomography, and a review of computerized transaxial tomography. Simple, reproducible radiographic tomographic methods are increasing in use, and despite their complexity, the diagnostic results are superior to conventional radiographs. There are many different motions of the X-ray tube and the film which can be employed to create a tomogram. The perfect tomographic motion is partly determined by the geometric shape of the object to be imaged and the thickness of the plane of interest. The undirectional tomographic method blurs a point in a linear fashion; the pleuridirectional method blurs a point over a wider surface. Among the most popular directions used are linear, circular, elliptical, and hypocycloidal. The numerous applications of tomography described in this review are an encouraging, broad-based foundation from which the most clinically useful and economically feasible devices will emerge.


Comparison of 85Sr, 87mSr, 18F, and 99mTc-labeled phosphates for bone scanning.

The observation by Subramanian and his co-workers that a 99mTc-labeled polyphosphate had excellent affinity for bone has led to widespread use of 99mTc-labeled phosphates as bone scanning agents. Initially, only polyphosphate was employed, but because of somewhat inconstant results and difficulty in preparation of this product, other phosphate compounds were sought. We soon discovered that an inorganic compound, pyrophosphate, appeared to have certain advantages over polyphosphate. Other workers formulated diphosphonates (organic phosphates) which also demonstrated advantages over polyphosphates. Comparison studies in rabbits utilizing 85Sr, 87mSr, 18F, and several phosphates (inorganic and organic) proved the 99mTc-labeled phosphates to be clearly superior in delineating normal skeletal anatomy. Studies in humans confirmed that excellent visualization of bone was obtained with 99mTc-labeled phosphates using either a gamma camera or a rectilinear scanner. What was not known, however, was just how reliable this class of agents would prove to be in detecting bone disease when compared to bone-seeking radiopharmaceuticals such as 85Sr, 87mSr, and 18F. Further comparative analyses have clearly demonstrated that both inorganic and organic 99mTc phosphate complexes are extremely sensitive in revealing more bone disease than the older bone scanning agents.


Biochemical anomalies of the nephrotic syndrome.

The nephrotic syndrome is the only hypoalbuminaemic state frequently associated with hyperlipidaemia. In the presence of a negative nitrogen balance, hyperlipidaemia is metabolically inappropriate and reflects the result of persistent breakaway from free fatty acid control. This lipid abnormality may result in the premature development of ischaemic heart disease in patients in whom it is not possible to control the primary renal abnormality. The authors suggest that future work should be directed towards thyroxine and insulin metabolism in nephrotic states.


Diagnostic fiberoptic bronchoscopy: Techniques and results of biopsy in 600 patients.

Six hundred patients underwent diagnostic flexible fiberoptic bronchoscopy (FFB). The two diseases most frequently encountered were bronchogenic carcinoma in 330 patients (55 percent) and bacterial infection in 94 (16 percent). A positive cytology on biopsy material was obtained in 279 of 330 patients (85 percent) with primary lung cancer. Fluoroscopy was a valuable aid in diagnosing bronchogenic carcinoma, since 42 percent of the tumors were not visible endoscopically and required fluoroscopic control for placement of the biopsy instrument. Of the 55 patients with hemoptysis and negative chest x-ray films, nine (15 percent) had fiberoptically visible endobronchial carcinomas! In addition, two patients with carcinoma of the larynx and one with carcinoma of the nasopharynx were discovered. Transbronchial biopsy (TBB) in 68 patinets with diffuse and localized disease achieved an overall 69 percent diagnostic success, including a correct diagnosis in each of four patients with Pneumocystis carinii pneumonia. Brush biopsy provided additional valuable laboratory data in bacterial, mycobacterial and cytomegalovirsu infectious but had a poor yield in Pneumocystis infection. Complications as a result of forceps biopsy were minimal, except for brisk bleeding in six patients.


Myocardial and plasma levels of adenosine 3':5'-cyclic phosphate. Studies in experimental myocardial ischemia.

Alterations in myocardial and plasma levels of adenosine 3':5'-cyclic phosphate (cyclic AMP) were studied following clamping of the aorta or coronary artery occlusion in 30 dogs. Plasma cyclic APM levels increased markedly after thoracotomy but returned to control levels two hours later. Complete arrest of aortic flow (clamping) induced a significant early increase in the myocardial cyclic AMP levels of all animals studied. No increase was noted following pretreatment with propranolol or sham-occlusion. After localized coronary occlusion, only modest and insignificant changes occurred in plasma cyclic AMP levels in anesthetized animals and also in conscious dogs. The present study suggests that adrenergically mediated changes in tissue cyclic AMP content are an early manifestation of both generalized and local myocardial ischemia, while the plasma cyclic AMP level is a relatively insensitive indicator of small coronary occlusions.


The current status of prostaglandins and the lungs.

The prostaglandins are a group of pharmacologically potent lipid compounds whose role in pulmonary pathophysiology remains to be defined. There is not question that these compounds can induce constriction (PGF) or relaxation (PGE) of the smooth muscle in bronchi and pulmonary vessels. There is no question that the lungs are a major site of both the synthesis and degradation. These facts make speculation about the role of the prostaglandins in pulmonary disorders a tempting exercise. However, the current paucity of clinical data prevents any valid assessment of such speculation at this time and emphasizes the need for continued investigation of the role of prostaglandins in patients with lung disease.


Asymptomatic pulmonary granular cell tumor presenting as a coin lesion.

Granular cell tumor is a rare, usually benign neoplasm of neurogenic origin first described in 1926. Only 46 lesions, including the present case, of the 500 reported cases of this tumor, originated within the tracheobronchial tree. The case reported here is distinctly unusual because of its asymptomatic presentation as a coin lesion on chest roentgenogram. Electron microscopic examination of the tumor is presented.