Nuclear DNA polymerases of human carcinomas.

Comparisons have been made of the DNA polymerases of normal human lung and cecum, primary carcinomas of human lung, breast, and cecum, and resting and regeneration rat liver. The picture for the normal human tissues is similar to the one for unstimulated rat liver, that for the human carcinomas resembles regenerating rat liver. The human tissues contain two polymerases with sedimentation coefficients of about 3 and 7 S, the enzymes are restricted to the nucleus, and the specific activities of the 7 S polymerase, but not of the 3 S enzyme, are elevated in the cancers. Just as with the regenerating rat liver polymerases, the 3 S activity of a bronchogenic carcinoma is unaffected by cytosine arabinoside 5'-triphosphate and only little reduced by novobiocin, whereas DNA synthesis by the 7 S enzyme is abolished by both compounds. A variety of other inhibitory agents have similar effects on the 7 S polymerases of the human carcinomas and regenerating rat liver.


Characterization of macrophage chemotaxins in tumor cell cultures and comparison with lymphocyte-derived chemotactic factors.

Culture fluids from five murine sarcomas were chemotactic for syngeneic peritoneal macrophages in vitro. Peritoneal macrophages from mice infected with Mycobacterium bovis, strain Bacillus Calmette-Guérin, were more responsive to the chemotactic factor in tumor cultures than were normal macrophages. Peritoneal granulocytes, however, did not significantly respond to this factor. The level of chemotactic activity in tumor cultures paralleled cell growth for all five tumors; maximal levels occurred during log growth. Culture medium alone or fluids from proliferating spleen cell cultures stimulated with mitogens did not have detectable chemotactic activity. Chromatography of the tumor culture fluids resulted in a single peak of chemotactic activity in the 15,000-molecular weight range on Sephadex G-100 and at about 7.5 mmho/cm specific conductance on diethylaminoethyl cellulose. By both biological and physicochemical characteristics, the chemotactic activity in tumor culture fluids was different from mouse lymphocyte-derived chemotactic factor.


Consequences of inhibition of guanine nucleotide synthesis by mycophenolic acid and virazole.

Mycophenolic acid and virazole are inhibitors of inosinate dehydrogenase and produce growth inhibition and loss of viability in cultured murine lymphoma L5178Y cells. Treatment with 1 muM mycophenolic acid produced the following changes in concentrations of acid-soluble nucleotides: (a) guanosine triphosphate decreased to less than 10% of control within 2 hr; (b) uridine triphosphate and cytidine triphosphate concentrations increased markedly; (c) adenosine triphosphate did not change; (d) deoxyguanosine triphosphate decreased; and (e) thymidine triphosphate increased. DNA synthesis was inhibited by 90% within 2 hr, whereas the incorporation of adenosine into RNA and of leucine into protein were much less affected. Virazole (100 muM) produces similar effects. These biochemical effects of mycophenolic acid, as well as its effects on cell growth, can be prevented by addition of guanylate to the medium. Mycophenolic acid treatment also appears to cause breakdown of high-molecular-weight DNA.


Casein production by human breast cancer.

Casein was measured in the sera of breast cancer patients, in breast cancer tumors, and in breast cancer cells in long-term tissue culture using a sensitive and specific radioimmunoassay. Levels present in breast cancer sera were not elevated above control values. Eight of forty-seven (17%) of the tumor samples tested were positive for casein, the highest level representing 0.003% of the soluble protein. When seven human breast cancer cell lines were assayed for casein, the results were uniformly negative even under conditions of stimulation by lactogenic hormones. In addition, direct immunoprecipitation of labeled cellular protein supported the negative result of the radioimmunoassay. Thus it appears that casein production is not a common characteristic of most human breast cancers.


Evidence for carrier-mediated transport of melphalan by L5178Y lymphoblasts in vitro.

Mechanism of transport of the alkylating agent [14C]melphalan was investigated in L5178Y lymphoblasts in vitro. A time course of melphalan uptake was approximately linear for 5 to 10 min and thereafter entered a plateau region. Evidence that unidirectional influx of melphalan is carrier mediated was that uptake obeyed simple Michaelis-Menten kinetics, that it demonstrated chemical specificity, and that the cell/medium distribution ratio of drug decreased with increasing extracellular drug concentration. The kinetic parameters for melphalan transport consisted of a Km (mean +/- S.E.) of 1.53 +/- 0.18 X 10(-4) M and a transport capacity (Vmax) of 3.48 +/- 0.31 X 10(-17) mole/min/cell. Findings suggesting that transport was at least in part energy dependent and not simply a passive process were that drug uptake was temperature sensitive and sodium dependent. Analysis of cell sap constituents indicated the presence of intact drug within the cell. The percentage of radioactivity (mean +/- S.D.) found in the cell sap fraction was 95.8 +/- 2.2% of total cell activity, and 92.6 +/- 4.1% of this was trichloroacetic acid soluble. Thin-layer chromatography of the cell sap fraction and medium each revealed that the majority of radioactivity migrated as a single peak with an RF value identical with that obtained for free drug. The alkylating potential of intact drug complicated interpretation of the finding of apparent uphill transport against a concentration gradient. This observation, together with the relatively low cell-medium ratio (mean +/- S.D.) of 3.07 +/- 1.07, favors the concept that melphalan transport occurs by a facilitated diffusion process, although an active transport system has not been entirely excluded. The relative insensitivity of melphalan uptake to a wide range of metabolic inhibitors also suggests that transport is by a facilitated diffusion mechanism rather than an active process. Other alkylating agents and several amino acids including the L and D isomers of phenylalanine did not inhibit melphalan transport; thus a native substrate was not identified for the melphalan carrier and transport was by a mechanism separate from that of other alkylating agents.


The glutathione S-transferases of the small intestine in the rat.

Glutathione S-transferase activities have been identified in the small intestine of the rat. Thrree activities obtained with p-nitrobenzyl chloride (aralkyl), 1,2-epoxy-3(p-nitrophenoxy)propane (epoxide), and ethacrynic acid (alkene) as substrates were present in significant amounts. Gel filtration indicated an elution volume for the intestinal transferase activities that was similar to those activities in the liver and kidney. The induction of the intestinal transferases by polycyclic aromatic hydrocarbons and phenobarbital is similar to those effects observed previously for the hepatic and renal enzymes. The highest concentration of transferase activities occurs in the proximal small intestine; these activities are reduced upon fasting. Parallel observations have been reported for aryl hydrocarbon hydroxylases. Because only low or negligible levels of epoxide hydrases have been reported in the small intestine, the glutathione S-transferases may be the primary epoxide-detoxifying system in that organ.


The phosphorylation of histones in rat liver undergoing malignant transformation.

The incorporation of inorganic phosphate into H1-histones of rat liver was stimulated after the repeated s.c. administration of diethylnitrosamine. This stimulation was observed as early as after the fifth daily injection of the carcinogen and amounts to approximately 3 times the control value on the 60th day of the experiment. The effect was reversible when the application of the carcinogen was not extended beyond 4 weeks. A correlation was observed between these phenomena and alterations in the morphological structure, although the latter required a higher amount of single doses before the first signs of a forthcoming malignant transformation were seen. There was a difference in distribution of phosphate between the individual phosphorylation sites of the H1 molecule as compared to stimulated phosphate incorporation induced by adenosine cyclic 3':5'-monophosphoric acid or liver regeneration. The stimulated phosphorylation was not due to the inflammatory action of the carcinogen.


In vitro and in vivo cytotoxicity of adriamycin and daunomycin for murine macrophages.

In virto exposure of murine lymphoma cells to adriamycin and daunomycin (DM) for 24 hr resulted in comparable cytotoxicity as assessed by viable cell counts and inhibition of DNA synthesis. The spontaneous DNA synthesis of spleen lymphocytes in vitro was also equally affected by the two agents. However, when the in vitro effects of the two drugs on macrophage monolayers were investigated, DM was found to be at least four times more toxic than AM. In vivo-administered DM was more suppressive than AM of Corynebacterium parvum-induced spleen macrophage cytotoxicity. The fact that DM is less effective than AM as a cancer chemotherapeutic agent in vivo may actually be due to the greater destruction of macrophages by DM and a resultant reduction in the contribution of host immunity to the anti-tumor action.


Morphology and serum dependence of cloned cell lines undergoing spontaneous malignant transformation in culture.

A number of morphological changes were found to correlate with the occurrence of spontaneous neoplastic transformation in sublines of five rigidly isolated clones of mouse embryo fibroblasts. These morphological changes included increased cytoplasmic basophilia, reduced spreading of cells on the substrate, increased nuclear:cytoplasmic ratio, greater heterogeneity in the size and shape of cells and nuclei, and more random orientation of cells. Because these changes were reproducible, occurring in some sublines of all five clones, they have been described and illustrated to serve as a guide for identifying spontaneous transformants among rodent fibroblasts in culture. Neoplastic transformation was determined by the growth of the cells as malignant neoplasms in syngeneic hosts. The spontaneous transformants, as compared with nonneoplastic fibroblasts derived from the same cell, showed similar saturation densities and serum dependence. Some clones had a higher transformation frequency than the parental line, which remained nonneoplastic for years. Thus, the capacity for continuous growth in vitro can be independent of malignant potential. The use of horse serum as supplement to the medium did not accelerate or increase the frequency of neoplastic transformation.


Topography of nonneoplastic and neoplastic cells of common origin.

The possibility that neoplastic transformation may characteristically alter cell surface morphology prompted a comparison by scanning electron microscopy of nonneoplastic and tumorigenic cell lines from a single clone of mouse embryo cells. Among those studied by scanning electron microscopy, six lines of this clone proved nonneoplastic, and nine others underwent neoplastic transformation in culture, as evidenced by tumor production in vivo. Combined cinephotomicrography and scanning electron microscopy allowed the determination of postmitotic time and topography of individual cells without perturbing the cells or detectably altering their surface morphology; no pattern of morphological change as a function of postmitotic time was evident in either nonneoplastic or neoplastic cell populations. Accordingly, these cell populations could be compared under their usual conditions of attached asynchronous growth despite differences in proliferation rates. Cells of the neoplastic lines were characteristically less spread, and some lines displayed greater morphological variability than was evident among cells of nonneoplastic lines. However, most cells in all nine neoplastic lines and all six nonneoplastic lines were smooth surfaced. Thus, the exaggerated incidence of microvilli, ruffles, or blebs reported for established tumor-derived lines and most morphologically transformed lines did not prove a reliable criterion of neoplastic state for these cell lines of common origin grown under the same culture conditions.


Identification and kinetics of G1 phase-confined cells in experimental mammary carcinomas.

Three lines of mouse mammary carcinoma growing in isogenic hosts and differing in degree of histological differentiation and rates of proliferation were used to study parenchymal cells with various types of nucleoli. The relative number of cells possessing trabeculate or ring-shaped nucleoli or nucleolar fragments was closely related to the growth rate and degree of differentiation of tumor lines tested. All three subpopulations increased with increasing age and with decelerated tumor growth. In some cells in late telophase, either trabeculate or ring-shaped nucleoli could be distinguished in mitotic poles. This demonstrated that cells with these nucleoli are detected at the beginning of G1 phase. Even low levels of DNA synthesis, which would indicate that some cells with trabeculate or ring-shaped nucleoli or possessing nucleolar fragments were in S phase, could not be demonstrated. Microfluorometric measurements have indicated that cells with trabeculate and ring-shaped nucleoli have a DNA content close to 2c, whereas cells with dense nucleoli have a DNA content corresponding to either 2c, 2 to 4c, 4c, or greater than 4c. On the basis of these observations, it is concluded that cells with trabeculate and ring-shaped nucleoli and cells with nucleolar fragments either proceed slowly through G1 or are arrested in this phase. Cells with trabeculate nucleoli were replaced steadily, having a transit time of no longer than 84 hr. These cells constituted a "fast" component of cell renewal of G1-confined cells. A "slow" component, cells bearing ring-shaped nucleoli or nucleolar fragments, were replaced after a lag of 24 to 48 hr. with residency time for some of these cells being in excess of 84 hr.


Time dependence of ethionine-induced changes in rat liver transfer RNA methylation.

Methyl-deficient transfer RNA (tRNA) and subnormal levels of tRNA-methylating enzymes were found in the livers of female rats that had received injections of 250 mg DL-ethionine per kg body weight per day and 120 mg adenine per kg body weight per day for 2 days. Adenine alone had no effect. When the ethionine plus adenine injections were continued for longer periods of time, liver tRNA-methylating enzyme activity measured in vitro gradually increased and exceeded that of the controls. Concurrently, the relative methyl deficiency of liver tRNA decreased. The latter was evident because of the decreased ability of the tRNA to accept methyl groups during in vitro methylation catalyzed by homologous enzymes. Liver tRNA from animals that were treated with ethionine for 7 days could accept only about 40% as many methyl groups as could tRNA from animals that had received ethionine for only 2 days. No further significant change in methyl deficiency of the tRNA was seen when ethionine administration was extended to a total of 14 days. Enzyme preparations from ethionine-treated, but not control, rat livers contained dialyzable substances that inhibited the tRNA methylases and altered the base specificity of these enzymes. Although S-adenosylhomocysteine and S-adenosylethionine were found to be present in the liver preparations, neither of these substances could account for the observed changes in specificity.


Tumor cure and cell survival after localized radiofrequency heating.

Radiofrequency electromagnetic fields at 13.56 MHz were used to heat locally EMT-6 sarcomas and KHJJ carcinomas in BALB/cKa mice. Temperature profiles obtained in tumors during treatment showed uniform temperature distribution throughout the tumor volume with no systemic hyperthermia. Temperature could be maintained at a stable level throughout treatment by adjustment of power. Tumors were treated at 43 degrees, 43-5 degrees, and 44 degrees, for 5, 10, 20, 30, and 40 min. The EMT-6 tumor was highly sensitive to cure by radiofrequency heating: a 5-min exposure at 44 degrees resulted in cure of almost 50% of the tumors. Cure rate was a function of temperature and of duration of exposure. The KHJJ carcinoma was somewhat more resistant to cure by radiofrequency heating, although most of the animals treated at 43.5 degrees or above were cured of their tumors. In an effort to explain the remarkable effectiveness of radiofrequency heating, tumor cell survival studies were done on EMT-6 tumors treated in situ. Cell inactivation by radiofrequency heating was similar to that for hot water bath heating. However, direct cell killing cannot account for the observed cures, and an additional mechanism must be responsible for tumor eradication.


Morphometric analysis of neoplastic transformation in rodent fibroblast cell lines.

Populations from seven pairs of neoplastic and nonneoplastic cell lines of common origin were compared for evidence of a change in morphotogical parameters accompanying neoplastic transformation. Projected cytoplasmic area, projected nuclear area, cell dry mass, nuclear dry mass, whole-cell absorption at 265 nm, and nuclear absorption at 265 nm were determined. Neoplastic transformation was consistently accompanied by three morphological features: (a) decrease in projected area of the lamellar cytoplasm; (b) decrease in the projected area of the nucleus; and (c) decrease in dry mass of the lamellar cytoplasm. Ultraviolet absorption was generally less in the neoplastic cells than in the nonneoplastic cells, while nuclear mass remained approximately the same. These results are interpreted as quantitative evidence that neoplastic transformation is accompanied by morphological change. The morphological events may be characterized as a loss in cell surface and nuclear membrane components.


Repair of ultraviolet light damage in a variety of human fibroblast cell strains.

Postreplication repair of DNA damage after ultraviolet light irradiation has been examined in a wide variety of human fibroblast strains. The donors were patients with xeroderma pigmentosum (XP) of different complementation groups or other hereditary disorders with indications of radiosensitivity, or with light sensitivity or multiple cancers. The defect in postreplication repair previously found in XP variants (excision-proficient XP's) has now been observed in a total of five XP variants and a less severe defect in postreplication repair has been found in excision-defective XP's in Complementation Groups A, B, C, and D. Complementation Group E and all other cell strains studied showed a response that was not significantly different from that of cells from normal donors. Excision repair was also measured in some of these cell strains and was found to be defective only in XP cells. Ultraviolet cell survival characteristics have been obtained for may of the cell strains. The most sensitive were cells from the excision-deficient XP's and from a sun-sensitive child (11961); the latter had no measurable defect in either excision or postreplication repair. The rest of the survival curves lay in a band limited by normal cell strains on the one hand and the slightly more sensitive excision-proficient XP variant XP30RO. Only in the case of the variants XP30RO and XP7TA were we able to demonstrate any influence of caffeine on cell survival.


Homologous neurosecretory cell groups in the land snail Achatina fulica and the sea slug Aplysia californica.

The right parietal ganglion of the African snail Achatina fulica contains an identifiable cluster of 20-40 white cells with somatic diameters of 80-110 mu. Cobalt chloride injection into these cells revealed an axon projection in the right parietal nerve as well as apparent terminations in the capsule. The cells contain an abundance of electron dense granules 1000-2000 A in diameter which are formed in the Golgi apparatus. The granules stain with Gomori's chrome hematoxylin and paraldehyde fuchsin methods. While most cluster cells showed no "spontaneous" electrical activity, there was a correlation for the active cells between rate of activity and intensity of cell whiteness. These and other data suggest that the cluster cells are neurosecretory. A claim of homology with the "rostral white cells", R3-R13, in the parieto-visceral ganglion of Aplysia is based on a similarity of morphological and electrophysiological features, together with analogous locations in the respective ganglia. It is concluded that the right parietal ganglion of Achatina is homologous with the right half of the parieto-visceral ganglion of Aplysia.


Ultrastructure of the rat pineal gland grafted under the kidney capsule.

Pineal glands were grafted under the kidney capsule of mature male rats for periods of 20, 40, 60 and 100 days. Each grafted gland was then excised and divided into two halves. One half was processed for conventional electron microscopy and the other was fixed in aldehydes and then incubated in a zinc-iodide-osmium tetroxide mixture at pH 4.4 (A-ZIO-4.4). During the forty days following the operation pinealocytes showed the typical ultrastructural features associated with cells with a high protein and/or peptide secretory activity. On the other hand, during this period, the number of granular vesicles decreased progressively. From day 40 on, the grafted pinealocytes lacked granular vesicles. During the second half of the experimental period the ultrastructure of the pinealocytes indicated that their secretory activity was considerably decreased. During the acute phase of the experimental period numerous structures regarded as the tip of growing axons as well as typical nerve fibres appeared around blood vessels and within the paren chyma of the grafted gland. In the transplanted tissue obtained 60 and 100 days after the operation the growth cones were scarce, whereas typical nerve endings became numerous. These endings contained small clear vesicles which reacted positively when the tissue was treated with A-ZIO-4.4. The secretory activity of the grafted pineal gland and the nature of the nerve fibres which innervate the graft are discussed.


Fine structure of the ordinary lateral line organ. II. The lateral line canal organ of spotted shark, Mustelus manazo.

The lateral line organ of the spotted shark is characterized by its semi-cylindrical shape. Each organ (neuromast) is so closely apposed to the next that the individual neuromasts are almost continuous. The neuromast is composed of receptor cells, supporting cells and mantle cells. The receptor cells bear one kinocilium and up to 40 stereocilia. Bi-directional arrangement of the receptor cells as occurs in teleosts was demonstrated. Afferent and efferent nerve endings were found at the base of the receptor cells. The supporting cells extend from the basal lamina to the free surface. Long microvilli and a cilium-like "ciliary rod" project from the top of each supporting cell. The cell contains relatively few elements of the Golgi apparatus and little rough endoplasmic reticulum, but mitochondria and filaments are abundant. The mantle cell limits the lateral margin of the neuromast. It is distinguished from the supporting cell because of its long crescent-shaped nucleus and scarce, short microvilli. Myelinated nerve fibres are found in the subepithelial connective tissue but not in the epithelium. The fine structure of the shark lateral line organ suggests that this organ is in an intermediated step of evolution between that of lamphrey and teleost.


Glycogen deposits in the pyloric gland of the ascidian Styela clava (Urochordata).

The pyloric gland of Styela clava contains large glycogen deposits that are digested by treatment with alpha amylase and depleted by 15 days starvation. The deposits are surrounded by cytoplasmic regions containing smooth endoplasmic reticulum and mitochondria. The cells also have rough endoplasmic reticulum, Golgi cisterns, lysosomes, microvilli, cilia, and lateral infoldings of the plasma membrane. The fine structure of the pyloric cells and the position of tubules between the absorptive epithelium and general circulation suggest that the gland functions as the vertebrate liver in carbohydrate metabolism. The pyloric cells of Styela do not appear to be excretory in a "renal" sense, since there is no infolding of the basal plasmalemma and mitochondria are usually associated only with the glycogen deposits. However, a hepatic-like excretory role is consistent with current findings. In light of the phylogenic affinities of vertebrates and ascidians, it is possible that the pyloric gland is homologous to the liver.


Neurosecretion in the basommatophoran snail Bulinus truncatus (Gastropoda, Pulmonata).

The neurosecretory system of the freshwater snail Bulinus truncatus was investigated. With the Alcian blue-Alcian yellow (AB/AY) staining method at least 10 different types of neurosecretory cells (NSC) were distinguished in the ganglia of the central nervous system. The differences in staining properties of the NSC - with AB/AY the cells take on different shades of green and yellow - are borne out at the ultrastructural level: the NSC types contain different types of neurosecretory elementary granules. The neurosecretory system of B. truncatus is compared to that of Lymnaea stagnalis, the species which has received the most attention among the pulmonates. It appears from the comparison that the systems of both species show many similarities, although some differences are also apparent.


Sertoli cells of adult rats "in vitro". A morphologic and functional study.

A cell line obtained from isolated seminiferous tubules of adult rat testis has been studied "in vitro" over a period of 35 days. Light and electron microscopic studies performed from hour 2 to the end of culture have shown the presence of a monomorphic cell population. After 5-6 days of culture the cells formed a monolayer. The cytoplasm of the cells contained numerous lipid bodies and produced numerous projections. The nucleus showed several indentations and one or more nucleoli. From the 9th to the 15th day of culture the cells developed a large amount of endoplasmic reticulum, Golgi apparatus and aggregates of electron dense granules. From the 20th to 40th day the cell cultures progressively degenerated. Immunochemical analysis of the culture medium revealed the presence of estradiol-17beta, which reached its maximum production rate from the 8th day to the 18th day of culture. Corresponding to cell involution estradiol concentration underwent a rapid decrease. On the basis of morphological and biochemical data the cells could be considered Sertoli cells.


Hypothalamic and hormonal control of photoperiodically induced vernal functions in the white-crowned sparrow, Zonotrichia leucophrys gambelii. 2. The effects of hypothalamic inplantation of testosterone propionate.

The effects of implantation of testosterone propionate (TP) in various sites in the hypothalamus on the photoperiodically induced vernal premigratory functions in the White-crowned Sparrows were investigated in order to assess the role of the hypothalamo-hypophysial-testicular axis in the induction of these responses. Implantation of glass capillary tubes containing TP in the basal infundibular nucleus (IN), in the median eminence, or in the pars distalis inhibited the photoperiodically induced increase in plasma levels of luteinizing hormone (LH), as measured by radioimmunoassay, and testicular growth. The effective inplants significantly lowered the levels of LH in birds held on nonstimulatory short days. These TP implants apparently inhibited release from the pars distalis of both LH and follicle-stimulating hormone (FSH). It is concluded that the site of sensitivity in the negative feedback by testosterone is either the basal IN or the pars distalis, or both. The implants of TP that inhibited the increase in plasma LH and testicular growth completely did not prevent the birds from fattening.


The villous stroma of the human placenta. I. The ultrastructure of fixed connective tissue cells.

In human placental villi the connective tissue is constructed by mesenchymal cells, small and large reticulum cells and fibroblasts. During early pregnancy mesenchymal cells dominate; starting with the third month of gestation the reticulum cells are in the majority within the terminal villi, the fibroblasts within the stem villi. Ultrastructurally intermediary types of cells can be differentiated. Together with reticular and collagenous fibres the reticulum cells form the basic architecture of the villous stroma during the first 2/3 of gestation: the "reticular type of stroma". This consists of a network of cells and fibres with fetal vessels fitted in between. The remaining interspaces form a fluid system of compartments in which Hofbauer cells are suspended. They are called stromal channels. During the last trimester these channels and the Hofbauer cells as well are progressively replaced either by voluminous masses of fibres ("fibrous type of stroma", mainly in the stem villi) or by sinusoidal enlargements of fetal capillaries ("sinusoidal type of stroma", mainly in the terminal villi).


Microtubules and extracellular microtubule-like structures in the retina of the rainbow trout.

In the retina of the rainbow trout (Salmo gairdneri, Richardson) two types of microtubular structures are demonstrated. Besides the normal type of microtubules (about 200 A in diameter), occurring in all cell types of the retina, a second type is described which is termed microtubule-like structure (MLS) because of its extracellular localization. These MLS have a diameter of about 250 A under the same preparative conditions in which the normal microtubules appear 180-200 A thick. The interspace between the tubules is smaller than between the microtubules. Specific MLS to membrane associations exist, which are analyzed by serial sectioning and tilting procedures. It is suggested that the MLS have their origin at small membranous extrusions of the plasmalemma. These extrusions could contain nucleation sites for the MLS-formation within the extracellular space. It remains unknown which cell type produces the MLS proteins and which factors are responsible for the aggregation of the subunits to intact MLS.


The cell cycle in the small intestine transplanted beneath the kidney capsule in syngenic mice.

Transplantation of a small fragment of the ileum beneath the kidney capsule in syngenic mice results in the formation of a cyst lined with proliferating intestinal epithelium. The duration of the cell cycle in the epithelium was determined (using tritiated thymidine and the FLM method) as 14.5 h, as compared with 11.5 h in the intestinal epithelium in situ. We conclude that the intestinal content has little effect on the cell cycle of epithelial cells of the small intestine.


Localization of vitamin A in the small intestine of mouse. An electron microscope radioautographic study.

Localization of vitamin A in the small intestine of mice was studied with electron microscope radioautography after administration of tritiated vitamin A. The label was concentrated over lipid droplets in cells distributed in the lamina propria and the submucous layer. The cells were similar both to fibroblasts and to fat-storing cells in their morphological features. The name "Vitamin A-Storing Cell" is proposed for these labeled cells, including the fat-storing cell in the liver.


Light microscope radioautographic study of glycoprotein secretion in the hypothalamic-neurohypophysial system of the rat, after L-fucose-3H injection.

Fucose-3H was injected into the cerebral ventricle of rats that were killed at several time intervals after injection. Semi-thin sections of the supraoptic nucleus and neurohypophysis were processed for radioautography and analysed quantitatively. Silver grains indicating the site of fucoselabeled glycoproteins were first located at the perinuclear region of the secretory neurons. The highest silver-grain density in these cells was observed at 2 h after injection , declining afterwards. Silver grains over the neurohypophysis were observed from 2 h on, reached a peak at 1 day after injection and decreased in the subsequent time intervals. The distributions of the silver grains over the neurohypophysis fitted Poissonian distributions and these were shown to be heterogeneous at the several time intervals. Pituicytes were not labeled. The percentage of silver grains over the Herring bodies increased with time. In rats deprived of water after fucose-3H injection there was a great increase in the release of labeled glycoproteins from the neurohypophysis. These results indicate that the glycoproteins synthesized by the secretory neurons of the hypothalamic nuclei are secreted in the neurohypophysis.


Ultrastructure of a scent scale organ with pressure discharge in male Caligo eurilochus brasiliensis (Fldr.) (Lepidoptera/ Brassolidae).

The abdominal scent apparatus of male Caligo eurilochus was examined at different ages by light, scanning and transmission electron microscopy. The glandular epithelium is covered with specialized scales and forms a pad on each side of the 4th to 6th abdominal segments. The pads are surrounded by smooth, elastic cuticle and can protrude toward the opposite hind wing hair pencil. The scales have a poreless cuticle with a fibrillar texture. They are impregnated by an oily, slowly volatile substance. The scales are elongated toward the base, forming hoods over the long cone-shaped sockets. The scale pedicel is anchored tension-free by rootlets in the central socket base. The slightly asymmetric cuticular sockets are very elastic, due to their high water content. They are stabilized by internal epicuticular rods. The release of the secretions from the cell and a possibly active microvillar transport is discussed. Different secretions are found in the space between the microvillar surface of the gland cell and the socket floor. They are probably discharged from the supraglandular space into the scale lumen by means of pressure and bending of the sockets. A flowback might be prevented by capillary effect of a "ball" of vesicles, which lies exactly above the outlet of the scale pedicel.


The superposition eye of Cloeon dipterum: the organization of the lamina ganglionaris.

The lamina ganglionaris of the superposition eye of Cloeon dipterum is composed of separate optic cartridges arranged in a hexagonal pattern. Each optic cartridge consists of one central, radially branched monopolar cell (Li) surrounded by a crown of seven retinula cell terminals and two more unilaterally branched monopolar cells (La1/La2) situated close together outside the cartridge. Projections to neighbouring cartridges have not been observed. In most cases, synaptic contacts could be seen between a presynaptic retinula cell and more than two other postsynaptic profiles, which belong to monopolar cells or sometimes to glial cells. Seven retinula cell fibers of one ommatidium pass in a bundle through the basement membrane, run into their respective cartridges without changing orientation and terminate at approximately equal levels in the lamina. Long visual fibers with endings in the medulla are not visible in the superposition eye lamina, but are present in the lateral apposition eye. The relationship between the behaviour of the animal, optic mechanisms of the superposition eye and the structure of the lamina is discussed.


Excitatory synapses of blue crab gastric mill muscles.

Physiological and ultrastructural studies were made of neuromuscular synapses in stomach muscles, especially two gastric mill muscles of the blue crab innervated by neurons of the stomatogastric ganglion. These muscles depolarized and contracted with application of glutamate, but not acetylcholine, whereas the dorsal dilator muscles of the pyloric region depolarized and contracted in acetylcholine, but not in glutamate. Large excitatory postsynaptic potentials (EPSP's) of 5-20 mV were recorded in the gastric mill muscles. At low frequencies of activation, individual synapses released on average about 2 quanta of transmitter for each nerve impulse. Facilitation of EPSP's after a single nerve impulse could be detected for at least 10 s. Synapses were found on enlarged terminals of the motor axon; their contact areas ranged from 0.2 mum2 up to 3mum2. Both electron-lucent, round synaptic vesicles and dense-cored vesicles occurred near these synapses. A possible correlation between contact area of a synapse and output of transmitter, is discussed.


The accessory outer segment of rods and cones in the retina of the guppy, Poecilia reticulata P. (Teleostei). An electron microscopical study.

The ultrastructure of the accessory outer segment (AOS)--a cilium-like structure emanating from the inner segment and running alongside the outer segment of photoreceptors--is described. The AOS occurs in both rods and cones of Poecilia reticulata. Its ultrastructure, including the arrangement of microtubules, which originate from the ciliary stalk, is the same in rods and cones. The cone-AOS is connected with the outer segment by a thin plasmabridge, whereaa the rod-AOS lies embedded within the outer segment. The outer segment of the cone, in contrast to that of the rod, is separated from the pigment epithelium by a large extracellular space. An intimate contact, however, is secured by the AOS; its membrane is closely appositioned to the pigment epithelium membrane. The functional significance of the AOS and its possible occurrence in other vertebrate classes, are discussed.


Ultrastructure of sea urchin tube feet. Evidence for connective tissue involvement in motor control.

An analysis of the ultrastructure of the tube feet of three species of sea urchins (Strongylocentrotus franciscanus, Arbacia lixula and Echinus esculentus) revealed that the smooth muscle, although known to be cholinoceptive, receives no motor innervation. The muscle fibers are attached to a double layer of circular and longitudinal connective tissue which surrounds the muscle layer and contains numerous bundles of collagen fibers. On its outside, the connective tissue cylinder is invested by a basal lamina of the outer epithelium to which numerous nerve terminals are attached. These are part of a nerve plexus which surrounds the connective tissue cylinder. The plexus itself is an extension of a longitudinal nerve that extends the whole length of the tube foot. It is composed of axons, but nerve cell bodies and synapses are conspicuously lacking, suggesting that the axons and terminals derive from cells of the radial nerve. Processes of the epithelial cells penetrate the nerve plexus and attach to the basal lamina. There is no evidence that the epithelial cells function as sensory cells. On the basis of supporting evidence it is suggested that the transmitter released by the nerve terminals diffuses to the muscle cells over a distance of several microns and in doing so affects the mechanical properties of the connective tissue.


Ultrastructural changes in midgut cells of female Aedes aegypti L. (Insecta, Diptera) after starvation or sugar diet.

The ultrastructure of the epithelial cells in the posterior part of the midgut in female Aedes aegypti was partly changed after starvation periods of 5 or 8 days. Most obvious is a drastic reduction of the amount of rough endoplasmic reticulum (rer), which is responsible for the synthesis of enzymes for blood digestion. A similar influence on rer membranes is to be observed in mosquitoes fed on sucrose solution only, without additional blood meals.


The effect of age on the ultrastructure of the mid-gut cells of the hymenopteran Nasonia vitripennis (Walk.) (Hymenoptera, Pteromalidae).

The ultrastructure of the mid-gut cells of aged female Nasonia vitripennis is described. The mid-gut is a shrunken and distorted organ in the aged animal. The individual cells are highly disorganised and the organelle components are altered. The small lipid droplets formed in the apical cell region do not coalesce to form the large central lipid inclusions characteristic of the young animal. The rough endoplasmic reticulum is reduced and some of the mitochondria enlarge. The mid- and apical cell regions also contain large numbers of cytolysosomes. The basal cell region is essentially unchanged, but the channels formed by the infolded basal plasma membranes are dilated. The changes observed are discussed in relation to previous observations on other insect species.


Neurosecretory system of the ventral ganglia in the dragonfly, Orthetrum chrysis (Selys) (Odonata: Libellulidae).

The neurosecretory cells of the ventral ganglia in the adult dragonfly, Orthetrum chrysis, are classified into A, B, C1 and C2 cells. The neurosecretory material in the ventral ganglia is composed of PAS-positive material with 1-, 2-glycol groups and some proteins. The proteins rich in cystine or cysteine occur predominantly in the A cells, moderately in C cells and negligibly in B cells. Proteins containing arginine occur in A and B cells only, and those containing basic amino acids occur in C2 cells. The neurosecretory pathways and the neurohemal organs are also described.


Structure and position of receptors within scales bordering the toes of gekkonids.

In gekkonids, the scales bordering the toes or the adjacent tissue possess suberpidermal and intraepithelial receptors in addition to setae-bearing organs. The position of subepidermal lamellated corpuscles seems to be correlated with the size of the species. The larger the adult animal the more frequently is this type of receptor found laterally in the toe. This can be explained in connection with the vibration-sensitive function of lamellated receptors. Intraepithelial axon terminals were found close to the setae-bearing sensilla in one species only. They are surrounded by numerous tonofibrils and may function as receptors for mechanical (pressure)-stimuli.


Electron microprobe investigations into the process of hard tissue formation.

The electron microprobe microanalyser has been used to measure the concentrations of Ca, P and S in the predentine of young rat incisors. The specimens were prepared as alcohol embedded ultrathin sections, unfixed vacuum embedded dry cut ultrathin sections and as thin cryostat sections. The results show the influence of preparation on the measured compositions and indicate that Ca is tightly bound to the matrix, whereas P can be easily washed out. Measurements along the dentine-predentine border demonstrated zones of Ca enrichment, the average size of which suggests that the zones could be the prestages of calcospherites. A mineralisation mechanism is discussed in which the high Ca concentration activates pyrophosphosphatase or ATPase before the onset of nucleation.


Radioautographic study of glycoprotein biosynthesis and renewal in the ovarian follicles of mice and the origin of the zona pellucida.

L-fucose-3H was injected intravenously into mice which were killed at several time intervals after injection and semi-thin sections of their ovaries were processed for radioautography and analysed quantitatively; At the same time the specific activity of serum glycoproteins was determined. Glycoprotein biosynthesis was demonstrated in the oocytes, granulosa and stromal cells. The silver grain density of the follicular fluid in large follicles reached a peak at 4 h, remained high at 8 h after injection and decreased steadily at the subsequent intervals. It was demonstrated that the labeling pattern of the follicular fluid depends on the secretory activity of the granulosa cells and also on the specific activity of serum glycoproteins. The collapsed zonae pellucidae which represent the highest degree of follicular atresia are able to take up glycoprotein macromolecules. Based on this finding and also on the labeling pattern of the large follicles it was shown that there is very little synthesis of specific glycoproteins for the zona pellucida in large follicles. A more specific labeling of the zona pellucida occurred in the medium follicles. Following the growth of these follicles having a previously labeled zona pellucida, it was demonstrated that this extracellular structure is secreted by the oocyte.


Ultrastructure of the epithelium from the ovary wall of Dendrochirus brachypterus (Pteroidae, Teleostei).

The inner epithelium of the ovary wall in Dendrochirus brachypterus does not take part in egg production, as it does in other teleosts. The epithelium consists of columnar cells rich in mitochondria and secretory organelles. The distal ends of these cells hang free in the ovary lumen, separated from each other and densely covered by large microvilli. During reproduction the epithelium secretes large amounts of mucus that forms an envelope around the eggs produced from a spongy stroma of the ovary, and keeps the spawn afloat for 24 h.


Quantitative electron microscopic analysis of the stratified epithelium of normal human buccal mucosa.

The epithelium of normal human buccal mucosa was subjected to stereologic analysis. Ten biopsies were selected from a total of 20 specimens collected from 10 to 15 year old females, and processed for light- and electron microscopy. At two levels of magnification, electron micrographs were sampled from four strata in epithelial ridges and from three strata in regions over connective tissue papillae. Stereologic point counting based on a recently improved system for analyzing stratified epithelia was employed to analyze a toatl of 1820 electron micrographs. Buccal epithelium was found to be 0.48 mm thick, interdigitated by long, slender connective tissue papillae, and comprised of a narrow basal and suprabasal, and a broad, homogeneously structured spinous and surface compartment. From basal to surface layers, the epithelium displayed a differentiation pattern different from that of keratinizing epithelia. This pattern was a function mainly of a drastic density increase of cytoplasmic filaments of a constant 80 A diameter, a corresponding decrease of the cytoplasmic ground substance, the appearance of dark-cored membrane coating granules and individually varying amounts of glycogen deposition. It is suggested that the dense meshwork of filaments which fill 70% of the epithelial cytoplasm in a broad subsurface and surface layer, serves as the functional matrix for epithelial distensibility.


On the ultrastructure of polypeptide hormone-producing cells in the gut of the ascidian, Ciona intestinalis L. and the bivalve, Mytilus edulis L.

Ultrastructural evidence has been found for the presence of polypeptide hormone-producing cells in the gut of Ciona intestinalis L. and Mytilus edulis L. which do not appear to have been described before. Due to their localization and ultrastructural characteristics, it is suggested that the cells in Mytilus edulis probably produce an insulin-like substance and that some of these cells in Ciona interstinalis may produce 5-HT (5-Hydroxytryptamine). In each species only one granulated cell type can be observed; The granules, which are electron dense and membrane bound, also show a halo. The average diameter of the granules is 100-200 nm for Ciona and 200-400 nm for Mytilus.


Freeze-fracturing of the auditory basilar papilla in the lizard Calotes versicolor.

Membrane-associated structures were studied in the cells of the basilar papilla, the hearing organ of a lizard, Calotes versicolor, by means of freeze-fracture. Zonulae occludents of an intermediate to tight type were found to join the apical membranes of sensory and supporting cells. Gap junctions were frequently observed between neighboring supporting cells. The size and shape of the gap junctions varied considerably. Their distribution was as follows: small junctions were seen apically, large ones basally. This finding suggests the existence of electrical coupling among the supporting cells. Only afferent synapses have been described in this organ. Aggregates of particles were found in the pre- as well as the post-synaptic membranes, similar to those described at synapses in the central nervous system.


Exocrine pancreas under experimental conditions. III. Membrane and cell junctions in isolated acinar cells.

The ultrastructure of the cell membrane and intercellular junctions was studied after isolation of exocrine pancreatic cells by tryptic digestion and mechanical treatment. The number and distribution of membrane associated particles does not change significantly when acinar cells in situ are compared to those after the isolation procedure. However, intercellular junctions undergo distinct alterations. Gap junctions in normal pancreatic cells are macular in shape and localized at the lateral parts of the cell membrane. In isolated acinar cells gap junctions are irregularly shaped, more extended, and frequently associated with tight junctions. Tight junctions form belt-like structures which are found to persist after isolation but subsequently become elongated and interrupted. Thus extensive macular areas of tight junctions develop. Further, the strands on the P-face and the grooves on the E-face of freeze-fracture replicas change in array, dissociate, and become loosely packed on large membrane areas. The present investigation shows that the intramembranous proteins of tight and gap junctions are mobile structures within the fluid membrane. The shape of their array is dependent on the form of the intercellular contact zone.


"Tight" junctions in the sheath of normal and regenerating motor nerves of the crayfish, Orconectes virilis.

"Tight" or occluding intercellular junctions occur between adjacent glial processes in normal and regenerating crayfish motor nerve sheaths. Although infrequent, these junctions possess the ridge and groove configuration characteristics of freeze-cleaved occluding junctions. When present, nerve sheath tight junctions consist of a single, or at most a few, parallel intramembrane ridges situated on the EF membrane face of the glial plasma membrane. Consequently, such contacts are rarely recognized in thin sections of plastic-embedded nerve sheaths. Crayfish nerve sheath tight junctions are of the fascia occludens type and, therefore, do not impede solute flow across the nerve sheath. Fasciae occludentes of regenerating nerve sheaths occur in close proximity to discoid plaque-like aggregates of particles assumed to represent maculae adhaerentes. This relationship, which was not observed in normal nerve sheaths, suggests a functional association between the two types of junctions, perhaps developmental transformation of one junction type into the other. Although ridges and grooves of tight junctions occur next to cross-fractured trans-glial channels, no functional significance is proposed for this relationship. This study is the first report of tight intercellular junctions in crustacean glial nerve sheaths.


Fine structure of the sensilla of Peripatopsis moseleyi (Onychophora).

Three types of sensilla occurring on the lips and on the antennae of Peripatopsis moseleyi have been investigated by scanning and transmission electron microscopy. On the lips sensory spines can be found which contain numerous cilia originating from bipolar receptor cells. They reach the tip of the spine where the cuticle is modified. The perikarya of the sensory cells, a large supporting cell with a complicated surface and a second type of receptor, form a bud-like structure and are surrounded by a layer of collagen fibrils. The second receptor cell bears apical stereocilia as well as a kinocilium which are directed towards the centre of the animal -- thus the cell appears to be turned upside down. The sensilla of the antennae are 1) sensory bristles containing two or three kinds of receptor cells, one of which bears an apical cilium and one kind of supportive cell and 2) sensory bulbs located within furrows consisting of receptor cells with branched cilia and two kinds of supportive cells which are covered by a modified thin cuticle. According to the electron microscopical findings the sensory spines on the lips are presumably chemoreceptors. The sensory bristles on the antennae can be regarded as mechanoreceptors and the sensory bulbs as chemoreceptors.


Cellular mechanisms of stimulation of bud production in Hydra by low levels of inorganic lead compounds.

Treatment of Hydra with subtoxic levels of inorganic lead compounds (lead nitrate and lead chloride) for perods ranging from 5 min to one hour causes a temporary increase in bud production as compared to untreated control animals. This effect can be inhibited by the addition of large amounts of calcium chloride to the culture medium. The increased rate of budding is preceded by a dramatic increase in the number of nerve cells per animal, which is first observed within six hours after lead treatment. This appears to be the result of an increased rate of mitosis in the undifferentiated interstitial cells and their subsequent differentiation into nerve cells. The total number of cells per animal also increases after exposure to lead compounds, suggesting that lead may act as a general mitotic stimulator of all dividing cell types in Hydra.


Formation of primordial yolk granules in the avia oocyte.

Electron and light microscopical investigations of early oocytes (between 1.0 mm and 5.0 mm in diameter) from the ovary of 28-30 week-old chickens, suggested the formation of primordial yolk granules from cytoplasmic vesicles. These vesicles formed an aggregation which was observed to be surrounded by membranes, giving the aggregate a multivesicular body-like appearance. At a later stage the vesicles inside the membrane disintegrated and the multivesicular bodies acquired the appearance of primordial yolk granules. The contribution of other structures to the formation of yolk granules is discussed.


The hypothalamo-choridal tract. II. Ultrastructural response of the choroid plexus to vasopressin.

Light and electron microsopic examination of choroid plexuses from lateral ventricles of water-deprived and subcutaneously or intravenously vasopressin administered rats reveal morphologic changes typical for vasopressin responsive fluid transporting epithelia during hormonal stimulation. Ultrastructural changes noted in both dehydrated and vasopressin treated animals included: the frequent occurrence of choroidal "dark" cells, dilatation of the lateral and basal intercellular spaces, increased vacuolization of the apical cytoplasm, and a change in microvillar conformation from the normal clavate type to those with a filiforma shape. On the basis of the ultrastructural changes observed it is proposed that the choroids plexus be regarded as a target tissue for vasopressin. These findings indicated that a vasopressin mediated transchoroidal cerebrospinal fluid absorption capability exists.


Endocrine-like cells of the pulmonary epithelium of the human adult lung.

The morphological and histochemical characteristics of endocrine-like cells of the pulmonary epithelium of the right lower lobe of 12 human adult lungs were studied. Few cells were reactive to the argyrophil silver method of Grimelius and of Sevier and Munger and cells with a similar morphology and distribution emitted a green or yellow fluorescence after treatment of the lung epithelium with the amine presursors L-DOPA or L-HTP, respectively. A greater number of cells seems to be demonstrated by electron microscopy. The cells were characterized by small, round secretory granules showing a central dense core and a very thin clear halo between the core and the surrounding membrane. The cells are thought to be related to the endocrine-like cells of the pulmonary epithelium of the human foetal lung and to cells of carcinoids of larger bronchi.


Measurements of noradrenaline fluorescence in axons of chick embryo sympathetic ganglia grown in three day culture.

Chicken embryo sympathetic ganglion fragments, grown in culture for three days, were incubated for short periods in appropriate experimental and control media to provide information on axonal uptake of exogenous noradrenaline in the presence and absence of the uptake-blocking drugs, protriptyline and phenoxybenzamine. The cultures were dried, gassed in formaldehyde vapour and examined by fluorescence microscopy. Spot readings were taken on axon-bundles using a microspectrophotometer. Readings were corrected for the amount of tissue present in each field, using graticule and interferometric methods. Brightness was uniform along individual axon-bundles, but varied from bundle to bundle within the same culture. Despite this range in brightness the readings fell into a normal distribution about the mean. Dose-dependent increases in brightness were obtained following incubation with noradrenaline. A clear indication of the effect of the blocking drugs was observed but the range in brightness prevented reliable quantitative data on these effects from being obtained. It was concluded that the wide range of brightness within each culture reflected a lack of equivalence in the intrinsic noradrenaline-uptake properties of the axon-bundles developing in such cultures.


The development of innervation in the rat atrioventricular node.

The problem of development of the innervation of the rat atrioventricular node has been investigated by electron microscopy. Nerve bundles appear in relation to the node as early as the second postnatal day and vesiculated axons are seen throughout the entire node by the fourth day. Intimate contacts between nodal cells, axons and terminal varicosities are frequently observed. Use of the 5-hydroxydopamine tracer technique has enabled the identification of both cholinergic and adrenergic axons. It is concluded that the node has a dual innervation although cholinergic endings far outnumber those classified as adrenergic on the sixth postnatal day. These results are quite different to earlier findings made at the light microscope level and the discrepancies are discussed with respect to the histochemical techniques used. The suggestion that nodal differentiation is induced by nerves is considered in relation to the differences in cholinesterase activity exhibited by nodal cells during normal development and following neonatal sympathectomy.


Specialization of the endoplasmic reticulum in the apex of the midgut cells of Aeshna cyanea (Insecta, Odonata).

Lamellar stacks of cisternae regularly spaced and bound to each other by fine filaments are observed in the apex of midgut cells of Aeshna cyanea. These cisternae are connected with the rough endoplasmic reticulum but are devoid of ribosomes except on their terminal sacs. Their location and their structure, quite different in fed and starved animals, suggest that they are involved in intracellular transport.


Incidence and age distribution of patients with myocardial infarction and normal coronary arteriograms.

Over a 41 month period selective coronary arteriography was performed on all patients age 35 and under seen at our hospital with a documented myocardial infarction. In these 25 patients, 4 (16%) demonstrated no arteriographic evidence of coronary artery disease. One-hundred and fifty-two patients over age 35 with a documented myocardial infarction underwent selective coronary arteriography during the same period. In each of the 15 2 cases, obstructive coronary artery disease was demonstrated. The generally favorable prognosis of patients with myocardial infarction and normal coronary arteriograms has been previously documented. On the basis of our experience and a review of the literature, it is recommended that all patients age 35 and under sustaining a myocardial infarction should undergo selective coronary arteriography, in order to establish prognosis.


A simplified method of echocardiographic data analysis.

Rapid accurate analysis of echocardiographic data is accomplished using a sonic digitizer and programmable calculator. This method allows the echocardiographer to select technically optimal areas of the recording for analysis. The resolution of the measuring device is 0.1 mm. A hardcopy printout of both measurement and calculation is provided. Instead of expensive on-line computer, an inexpensive programmable calculator is used.


Pulmonary artery wedge pressures in congenital heart disease.

The bilateral measurement of pulmonary artery wedge pressure is essential in the cardiac catheterization evaluation of all patients with pulmonary artery hypertension. Five cases of pulmonary venous obstruction are presented, 4 of whom had additional intracardiac defects. The pulmonary artery hypertension in these 4 patients was initially attributed to the associated cardiac anomalies and because the left atrium had been entered directly with the catheter in each case, pulmonary artery wedge to the left atrium pressure gradients were initially either not obtained or were discounted as being artificial. Pulmonary venous obstruction was recognized in all 5 cases on the basis of an elevated pulmonary artery wedge pressure, and the anatomic site of the obstruction was successfully documented. Unless bilateral pulmonary artery wedge pressures are measured in all patients with pulmonary artery hypertension regardless of the presence of additional cardiac anomalies, a surgically correctable cause of pulmonary hypertension may be overlooked.


Characterization of left ventricular external wall motion in man by video dimension analyzer (Vidian).

Several investigators have described close relationships between left ventricular wall motion and physiologic cardiac events. Using an improved wall motion tracking devide (Vidian) in studies of 30 patients, we have compared the dynamics of left ventricular wall motion, recorded noninvasively, with high fidelity left ventricular and aortic pressures, intracardiac phonocardiograms, apexcardiograms, and cyclic left ventricular volume curves obtained during cardiac catheterization. Wall motion tracking signals comprised: pre-ejection outward deflection commencing with the first component of the first heart sound and coincident with the pre-ejection phase of the left ventricular pressure and apexcardiogram; a sharp descent during ejection, commencing with the "E" point of the apexcardiogram and with the onset of the upstroke of the aortic pressure; end ejection nadir, synchronous with the dicrotic notch of the aortic pressure; a nadir representing cessation of inward displacement, presumably reflecting slight inertial motion of the wall; a brief period of isovolumic relaxation which terminated synchronously with the "O" point of the apexcardiogram; rapid, then slow filling waves, coincident with those of the apexcardiogram, and demarcated by a transitional angulation synchronous with the third heart sound; and "a" wave, occurring simultaneously with that of the apexcardiogram. Ventricular wall motion tracking signals also corresponded to curves representing cyclic changes in left ventricular minor radius, and chamber volume derived from cineventriculograms. In 10 patients with abnormal contraction patterns detected by biplane cineventriculography, anomalous deflections were also recorded during ejection by the Vidian. Left ventricular wall motion tracking with the Vidian: 1) provides a sensitive index for timing of intracardiac events, 2) reflects cyclic changes in ventricular volumes and minor dimensions, 3) provides a convenient noninvasive technique for detection of regional asynergy involving the lateral left ventricular wall, and 4) by correlation with simultaneous ventricular pressure measurements, may provide useful information regarding left ventricular pressure/segment dimension relations.


External counterpulsation: coronary hemodynamics and use in treatment of patients with stable angina pectoris.

External pressure counterpulsation (ECP) has been reported to improve the clinical status of patients with angina pectoris. To document the mechanisms for such an improvement left ventricular oxygen consumption and lactate metabolism, coronary sinus blood flow, and cardiac index were studied in 10 patients with angina pectoris 1) prior to and during ECP; and 2) during right atrial pacing before and after 4 consecutive 2-hour sessions of ECP treatment. During ECP peak early and mean arterial diastolic pressures were significantly raised above control values by 32 and 13% respectively. However, coronary sinus blood flow, left ventricular oxygen consumption and left ventricular lactate extraction, mean systolic arterial pressure and cardiac index were not significantly altered by ECP. Right atrial pacing at 140 beats/min increased coronary sinus blood flow 70% over control values and induced angina and ischemic ST segment changes in 8 patients before and after 4 consecutive treatments of ECP. ECP treatment did not significantly modify the above metabolic and hemodynamic responses at rest or during atrial pacing. Although 5 patients reported improvement in angina symptoms the effect was transitory. No significant improvement over pre ECP-treatment exercise angina threshold was observed immediately following or at 1 and 3 months post treatment. This method of noninvasive circulatory assistance appears to be of doubtful value in the management of patients with stable angina pectoris.


Effects of propranolol on coronary hemodynamic and metabolic responses to tachycardia stress in patients with and without coronary disease.

To clarify the influence of propranolol-and particularly its heart-rate effects-on myocardial ischemia, coronary hemodynamics and metabolism were studied in 15 patients utilizing a protocol to control heart rate. Ten patients had significant coronary narrowing (CAD) and 5 were normal. Systemic pressure, coronary sinus blood flow (CSBF), left ventricular oxygen utilization (LVVO2), ST Segment depression, and myocardial lactate extraction were measured before and after propranolol (10 mg IV), at rest, during pacing-induced tachycardia stress. Propranolol-related reduction in CSBF and LVVO2 at rest was reversed when heart rate was controlled in both patient groups. Propranolol failed to alter heart-rate threshold, tension-time index (TTI), CSBF, or LVVO2 at angina in the CAD patients. Likewise, ischemic-type ST depression, decreases in lactate extraction, and coronary resistance were unchanged compared to values observed during tachycardia stress before propranolol. In normal coronary patients, propranolol also produced no significant change in LVVO2 or coronary resistance when its heart rate effects were controlled. These data imply that a major coronary and metabolic influence of propranolol relates to changes occurring secondary to its influence on heart rate. Furthermore, this agent's anti-ischemic effect is not prominent during tachycardia stress suggesting that this stress test may be clinically useful in patients taking propranolol.


Single left pulmonary vein with normal pulmonary venous drainage: association with partial anomalous pulmonary venous return.

An unusual case of a single left pulmonary vein draining the left lung and joining the left atrium without venous obstruction is reported. This anomaly occurred in association with partial anomalous pulmonary venous return from the upper lobe of the right lung to the superior vena cava. A similar case has been previously reported from our laboratory. The diagnosis of a single left pulmonary vein can be made by routine chest roentgenography because of the characteristic radiologic appearance of this anomaly. It is important to distinguish this benign entity from more ominous pulmonary radiodensities.


Long-term survival in preductal atresia of the aortic arch.

Atresia of the aortic arch is distinguished from complete interruption of the aorta by the presence of a fibrotic strand bridging the 2 discontinuous segments of aorta. Most patients with aortic arch atresia have associated intracardiac defects. There is a high neonatal mortality in this condition, with 95% of the patients dying in the first year of life. The case described herein is the first long-term survivor with atresia of the aortic arch, a patent ductus arteriosus, and an atrial septal defect to reach adulthood. In this patient an extensive collateral network existed in spite of the presence of a patent ductus arteriosus.


Determination of shunt flow in a case of multiple arteriosystemic connections.

A 64-year-old woman presented with acute congestive heart failure and atrial fibrillation, with good response to medical management. Selective coronary and left ventricular cineangiography demonstrated multiple arteriosystemic connections arising from both left and right coronary arteries, but normal (although tortuous) coronary vessels and ventricular contraction pattern. Determination of the magnitude of the shunt flow and concommitant measurement of myocardial lactate extraction suggested that this type of coronary vascular abnormality was an angiographic curiosity without discernible clinical import; however, the etiology of the patient's heart disease remains an enigma.


Coronary arteriography by the percutaneous transfemoral technique in patients on intraaortic counterpulsation.

Presented are the results of the use of the transfemoral approach for coronary arteriography, as a safe alternative to the brachial approach, in evaluating patients in cardiogenic shock. Five patients, each with an intraaortic balloon pump device in operation, were evaluated. No complications were encountered using the transfemoral approach.


Selective cannulation of the anterior interventricular vein and coronary sinus: potential applications.

A technique of cannulation of the anterior interventricular vein (AIV) selectively, along with selective cannulation of the coronary sinus, is described. The technique was successful in 61 of 82 patients (76%) in whom it was attempted. There were no serious complications. The technique allows one to obtain blood samples simultaneously from the anterior interventricular vein and the coronary sinus, so that the metabolism of the left ventricular anterior wall and that of the overall left ventricle can be studied. Possible clinical applications of simultaneous investigation of the left ventricular anterior wall and global myocardial metabolism are discussed.


Integrated viral sequences in adenovirus type 12-transformed hamster cells.

The physical state of the viral genome in four lines of hamster cells transformed by adenovirus type 12 (Ad12) has been investigated. The four lines of transformed cells originated from hamster cells after infection with Ad12 at multiplicities ranging from 5-350 plaque-forming units per cell. The DNA from transformed cells has been restricted with the Sal I endonuclease from Streptomyces albus which cleaves adenovirus DNA more frequently than DNA from adenovirus-transformed hamster cells. Thus after cleavage by the Sal I enzyme, it is possible to separate free adenovirus DNA sequences from these which are covalently linked to cellular DNA in transformed hamster cells. The results of sequential hybridization experiments in which the Sal I-treated DNA from transformed cells is first annealed to Ad12 DNA on filters, then eluted, and finally hybridized to hamster cell DNA, support the model of Ad12 DNA integrated in multiple fragments into the host genome. Further experiments will be required to characterize the host sequences adjacent to adenovirus DNA and to compare these sequences in different lines of Ad12 transformed cells.


Histone mRNAs contain blocked and methylated 5' terminal sequences but lack methylated nucleosides at internal positions.

Histone mRNA, labeled with 32P or 3H-methionine during the S phase of partially synchronized HeLa cells, was isolated from the polyribosomes and purified as a "9S" component by sucrose gradient sedimentation. We identified two types of 5' terminals, m7G(5')pppNmpN and m7G(5')pppNm-pNmpN, in which the first methylated nucleoside is 7-methylguanosine, the second is either N6, 2'-O-dimethyladenosine, 2'-O-methyladenosine, or 2'-O-methylguanosine, and the third is 2'-O-methyluridine, 2'-O-methylcytidine, or 2'-O-methyladenosine. Approximately 1.7% of the 32P label was present in the 5' terminal structures. Assuming a similar specific radioactivity for all phosphates, this percentage corresponds to an average of one terminal per 335 nucleotides. Histone mRNA differed from bulk polyadenylylated mRNA of HeLa cells in lacking significant amounts of 2'-O-methyluridine or 2'-O-methylcytidine in the second position of the 5' terminal oligonucleotide and in lacking N6-methyladenosine residues at internal positions.


Development of methods for the quantitative in vitro analysis of androgen-dependent and autonomous Shionogi carcinoma 115 cells.

Androgen-dependent and androgen-independent (autonomous), cloned, cultured cell lines of the androgen-dependent mouse mammary adenocarcinoma, Shionogi carcinoma 115, have been established. Growth of the dependent cells requires the presence of androgen, provided they are growth in suspension culture in medium containing dextran-charcoal-treated fetal calf serum. The growth rate of autonomous cells in the presence or absence of DHT is similar to that of dependent cells grown in its presence. An agar culture method has been developed that enables the proportion of dependent and autonomous cells in mixed populations to be determined. Autonomous cells appear in dependent clones, and their frequency increases with increasing time of subculture. Dependent cells form tumors preferentially in male animals and dependent cell cytosols contain significant amounts (approximately 300 femtomoles per mg protein) of a specific androgen-binding macromolecule. Autonomous cells formed tumors equally well in both male and female mice, and autonomous cell cytols contain very low levels (less than or equal to 7 femtomoles per mg protein) of the specific androgen-binding macromolecule(s). These studies delineate a system which can be used to investigate the mechanism of steroid hormone-dependent and autonomous tumor growth, and the transitions between the hormone-dependent and autonomous states.


Regulation of RNA polymerase II activity in alpha-amanitin-resistant CHO hybrid cells.

CHO hybrid cell lines obtained by fusing cells of wild-type sensitivity to alpha-amanitin with mutant cells containing RNA polymerase II activity resistant to alpha-amanitin have both sensitive (wild-type) and resistant forms of RNA polymerase II. When these hybrids were grown in medium containing alpha-amanitin, the sensitive form of polymerase II was inactivated, and the activity resistant to alpha-amanitin increased proportionally. The total polymerase II activity level therefore remained constant. This regulation of RNA polymerase II activity occurred independently of that of RNA polymerase I and was similar to that observed previously in the alpha-amanitin-resistant rat myoblast mutant clone Ama102 (Somers, Pearson, and Ingles, 1975a). A sensitive radioimmunoassay was developed to quantitate the total mass of RNA polymerase II enzyme. Under conditions of regulation of the enzymatic activity when hybrids grown in alpha-amanitin exhibited a 2-3 fold increase in the activity of the alpha-amanitin-resistant enzyme, no major change in the enzyme mass was detected immunologically. However, quantitation of the alpha-amanitin-inactivated polymerase II of wild-type sensitivity by 3H-amanitin binding indicated that the loss of its enzymic activity was accompanied by a loss of 3H-amanitin binding capacity in the cell lysates. All these results taken together indicate that a mechanism for regulating the intracellular level of RNA polymerase II exists and that it involves changes in the concentration of enzyme.


The molecular basis of emetine resistance in Chinese hamster ovary cells: alteration in the 40S ribosomal subunit.

The molecular basis of resistance to the protein synthesis inhibitor emetine has been examined in cell-free, protein-synthesizing extracts derived from normal and emetine-resistant (EmtR) mutants. We had earlier shown that protein synthesis in extracts of the mutant cells was resistant to the inhibitory action of emetin. When extracts from a wild-type and mutant cell line were fractionated into supernatant (S-100) and polyribosome fractions and mixed in different combinations, resistance to emetine was found to be associated with the mutant polyribosome fraction. Further fractionation of wild-type and mutant polyribosomes into 40S and 60S ribosomal subunits and mixing them in various combinations with an S-100 fraction from the wild-type cell indicates that resistance of mutant cells to emetine involves an alteration in the 40S ribosomal subunit. The behavior of EmtR has also been examined in somatic cell hybrids. Studies of EmtR X EmtS hybrid cell lines in vivo and in vitro show that EmtR is pheontypically recessive to EmtS, which is consistent with the ribosomal location of the genetic change.


A cytoskeletal structure with associated polyribosomes obtained from HeLa cells.

A method is described by which HeLa cells can be fractionated to reveal a skeletal-like structure in the cytoplasm. This cytoskeleton has many of the cell's ultrastructural features, such as 100A filaments, microfilaments, centrioles, and microspikes, although most of the cellular protein, membranes, and microtubules have been extracted. Associated with the cytoskeleton are most of the polysomal, but not the monomeric, ribosomes. These polysomes are distributed throughout the cytoskeleton except in the region of the 100A filaments, which resembles the distribution in intact cells. Degradation of mRNA with low levels of ribonuclease releases most ribosomes from the cytoskeleton. Prior disaggregation of polyribosomes in vivo releases ribosomes but not mRNA. Cytochalasin B administered in vivo releases the mRNA from the cytoskeleton. These results suggest an attachment of polyribosomes to the cytoskeleton via mRNA.


Visualization and mapping of late nuclear adenovirus RNA.

Nuclei of KB cells harvested at late stages of productive infection with adenovirus type 2 (Ad2) harbor RNA molecules which measure up to 13 mum in length, as determined by electron microscopy of denatured RNA. While some of the molecules display features of secondary structure that are characteristic for precursor rRNA, our interest was in those showing almost no intramolecular folding. When hybridized to double stranded viral DNA under conditions which favor RNA:DNA duplex formation, nuclear AD2 RNA displaces the homologous DNA region and generates R loop structures whose size is proportional to the length of the hybridizing RNA. Slowly sedimenting RNA forms small R loops, whereas RNA of high sedimentation velocity generates loops that span a large proportion of the DNA length. Using SV40 sequences within Ad2+ND4 hybrid DNA as a position marker, we oriented many of the R loops on the conventional Ad2 map. Our analysis was restricted to the most abundant sequences of late Ad2 nuclear RNA participating in R loop formation. A small but significant proportion of large RNA generates loops between map positions 0.3 and 0.9. The much more frequent RNA of intermediate size (although larger than mRNA) hybridizes with midpoints near map positions 0.55 and 0.88--that is, near the gene locations for hexon and fiber. Our findings are compatible with the idea that the nuclear RNAs visualized in this study are intermediates in a processing pathway leading to mature forms of late Ad2 mRNA.


How a single Sindbis virus mRNA directs the synthesis of one soluble protein and two integral membrane glycoproteins.

Previous work has shown that the 26S RNA found in Sindbis-infected chicken embryo fibroblasts encodes the three viral structural proteins, one internal protein, core, and two membrane glycoproteins, E1 and E2. This mRNA has one initiation site; core, E1, and E2 are derived by proteolytic cleavage. Here we show that during infection, the 26S RNA is found mainly in membrane-bound polysomes which synthesize all three virion structural proteins. These polysomes are released from the membrane upon treatment with puromycin and high salt. Newly synthesized core protein is localized on the cytoplasmic side of endoplasmic reticulum membranes, while newly synthesized envelope proteins are sequestered by the lipid bilayer. These results suggest that the nascent glycoproteins, presumably their amino termini, are of major importance in directing the binding of polysomes containing 26S mRNA to endoplasmic reticulum membranes and the subsequent transfer of glycoproteins into the bilayer.


Messenger RNA for myosin polypeptides: isolation from single myogenic cell cultures.

Messenger RNA which stimilates the synthesis of myosin heavy chain in a reticulocyte lysate has been isolated from single myogenic cell cultures. Specific myosin polypeptides have been identified by immunoprecipitation with an antibody made to purified adult chicken skeletal muscle myosin. This mRNA binds to oligo(dT)-cellulose, and an active fraction from sucrose gradients migrates as 26S on formamide-polyacrylamide gels. The relative amount of this RNA increases dramatically at the time of terminal differentiation.


Procollagen and collagen produced by a teratocarcinoma-derived cell line, TSD4: evidence for a new molecular form of collagen.

Procollagen and collagen were isolated from the culture medium and cell layer of line TSD4 (obtained from mouse teratocarcinoma OTT6050). SDS-polyacrylamide gel electrophoresis of the highly purified procollagen fraction demonstrated that the fraction is composed of theta chains (150,000 daltons), pro alpha chains (130,000 daltons), and alpha chains (100,000 daltons). Limited pepsin digestion of this fraction yielded a single species of collagen molecules having a chain composition (alpha1)3, as did collagen isolated from the cell layer. Each alpha1 chain appears to be slightly larger than alpha1 chains from calf or human type I and type III collagen. Amino acid analysis and cyanogen bromide peptide profiles of pepsin-treated TSD4 collagen demonstrated significant differences from those of other collagens (II, III, IV) of the type alpha1(X)3, although similar to that of the alpha1 chain of type I collagen, [alpha1(1)]2alpha2. Taken together, acrylamide gel electrophoresis, amino acid composition, electron microscopy, and cyanogen bromide peptide analysis indicate that this material represents a new molecular species of collagen not previously characterized, probably related to [alpha1(I)]3.


Clonal variation in colony morphology and growth of CHO cells cultured on agar.

Single Chinese hamster ovary (CHO) cells plated on agar form macroscopic colonies with high efficiency. Colonies produced by cells from the uncloned cell line increase in diameter continuously for 10-12 days after plating to form mounds of cells about 1 mm in diameter. With further incubation, some of these colonies do not increase in diameter (arrested dome), some form an expanding annular monolayer of cells around the central mount (fried egg), and some grow by enlarging the central mound into a low multilayered disc (saucer). These colony types on agar appear to be clonal characteristics of the CHO cell line. Cloning the line gives two kinds of isolates: one forms a mixture of arrested dome and fried egg colonies in an inheritable ratio, and the other forms saucer colonies. Cells from saucer colonies form saucer colonies when replated on agar. Cells from all colony types replate with similar efficiency on plastic or agar, and exhibit the same growth rate and cell size in liquid suspension culture. On plastic substrate, all these CHO cells form colonies which increase continuously in diameter for as long as 21 days, and little clonal difference in the morphology of colonies or of single cells is observed. These observations reveal a previously unsuspected heterogeneity in an established line of cultured mammalian cells and provide a method for studying new classes of in vitro growth control phenomena. These control phenomena may help in the building an in vitro model for tumor growth.


Metallothionein synthesis as an indication of the parenchymal origin of cells cultured from liver.

A line of cell derived from pig liver has been shown to retain the ability to synthesize zinc-thionein and to respond to Cd2+ with a greatly increased metallothionein synthesis, a property specific for liver and kidney parenchymal cells.


Heavy metal complexation in polluted molluscs. I. Limpets (Patella vulgata and Patella intermedia).

The accumulation of cadmium, zinc and copper by the marine gastropod molluscs Patella vulgata and Patella intermedia has been studied by gel permeation chromatography of water-soluble extracts of environmentally contaminated shellfish. A major proportion of the water-soluble cadmium and copper in these molluscs is associated with a protein of molecular weight 10 800 daltons. Evidence is presented supporting the similarity of this protein with mammalian metallothionein. This protein contained only a small proportion of the zinc found in the samples.


The in vitro oxidative metabolism of benzo(a)pyrene in human liver measured by different assays.

The specific metabolism of benzo(a)pyrene (BP) in adult human liver samples was studied by using triated BP and thin-layer chromatographic separation of metabolites and by using the conventional fluorometric assay. The incubation in vitro of BP with human liver homogenates yielded the usual pattern of metabolites, including several dihydrodiols, phenols and quinones, as well as products bound convalently to protein. Interindividual variation in the production of different metabolites were very large, several ten-fold. The over-all metabolism was inhibited by aminopyrine and SKF 525A but not by 7,8-benzoflavone. The formation of dihydrodiols was inhibited by several epoxide hydratase inhibitors with consequent accumulation of phenols and an unidentified metabolite (possibly BP 4,5-oxide). Radiometric measurement of phenols, total dihydrodiols and individual dihydrodiols correlated well with the fluorometric assay of BP hydroxylase activity.


Studies on the structure--activity relationship among aliphatic and aromatic bisguanylhydrazones and some related compounds.

A total of 18 compounds consisting of 7 alphatic and 7 aromatic bis(guanylhydrazones), p-quinone-bis(guanylhydrazone), one monoguanylhydrazone, one diamidine and one diguanidine were studied spectrophotometrically to determine their ability to interact with native calf-thymus DNA and the possible correlation of binding with biological activity. In each case, the ability of a compound to bind to DNA correlate with its ability to inhibit the activity of DNA-dependent DNA polymerase (EC 2.7.7.7) extracted from mouse leukemia L1210 cells. For example, all the aromatic bis-guanylhydrazones and diamidine (hydroxystilbamidine), which were good inhibitors of the enzyme activity, showed a biphasic interaction with DNA. All the aliphatic compounds displayed no detectable interaction with DNA in the Tris buffer used, and were also poor inhibitors of the polymerase activity. Interaction of decamethylene diguanide (Synthalin with DNA could not be determined because the compound does not absorb light in the UV-VIS region. However, in similarity with other aliphatic compounds, this agent was a poor inhibitor of DNA polymerase reduction. The p-quinone-bis(guanyl-hydrazone) and p-phenylbenzaldehyde-monoguanylhydrazone showed only a monophasic interaction with DNA and caused an intermediate inhibition of the enzyme activity. When tested for possible anti-leukemic activity against i.p. L1210 leukemia in syngeneic DBA/2J mice, all the aromatic bis-guanylhydrazones as well as hydroxystilbamidine caused prolongation of survival of tumor-bearing mice. Among the aliphatic bisguanylhydrazones, all of which showed no binding to DNA and caused at the most only a very slight inhibition of DNA polymerase, only methylglyoxal-bis(guanylhydrazone) (CH3--G) had antileukemic activity. Synthalin also inhibited leukemia growth. Evidences presented indicate that the mechanisms of action of aliphatic and aromatic bisguanylhydrazones may be quite different. Furthermore, the ability to bind to DNA may be a useful criterion to predict the antileukemic activity of aromatic guanylhydrazones and possibly other aromatic-bis-cationic compounds, but not that of aliphatic congeners.


Trypan blue: identification and teratogenic and oncogenic activities of its coloured constituents.

Three coloured substances frequently present as contaminants in commercial samples of trypan blue have been identified as those monoazo dyes in which 4-amino-3,3'-dimethyl-biphenyl, 4-amino-3,3'-dimethyl-4'-hydroxy-biphenyl or omicroc-tolidine are coupled to H-acid. These dyes have been synthesized and, together with purified samples of trypan blue, tested for teratogenic activity in mice and oncogenic activity in rats. Unpurified trypan blue was both teratogenic and oncogenic; purified trypan blue, was teratogenic but only weakly oncogenic; the monoazo dyes possessed neither activity. It is concluded that the main blue component of trypan blue is the teratogenic principle and that some as yet unidentified component of the purple fraction either is the main oncogenic principle or potentiates the action of the blue component.


Action of 1,10-phenanthroline transition metal chelates on P388 mouse lymphocyte leukaemic cells.

(1)Fully coordinated 1,10-phenanthroline and 2,2'-bipyridine chelates of Ru(II) are lethal in vitro to cultured and ascites P388 mouse lymphocytic leukaemic cells; 1,10-phenanthroline chelates are generally more potent than corresponding 2,2'-bipyridine compounds, and mixed-ligand (acetylacetonato) monovalent chelates of both series are more active than the corresponding identical-ligand divalent chelates. Lethal potency is greatest for Ru(II) chelates containing highly alkylated ligands. (2) Within two series of tetramethyl-1,10-phenanthroline chelates, the inert Ru(II) and Ni(II) members are less active against P388 cultured and ascites cells than the corresponding more labile chelates of Cu(II), Cd(II), Zn(II), Fe(II), and Co(II); for the ascites cells, the rank order of lethal potency of the chelates correlates reasonably well with their anticipated rank order of kinetic reactivity. (3) Repeated subculture of P388 cells in the presence of a mixed-ligand Ru(II) chelate has produced a cell line that shows a stable 10-fold resistance to the chelate; the resistant cell line is selectively cross-resistant to certain Ru(II) identical and mixed-ligand chelates. (4) The presence of a fluorescent Ru(II) chelate has been demonstrated at the surface and within the cytoplasm and nucleus of P388 ascites cells exposed to it either in vitro or in the mouse. (5) Ru(II) and Cu(II) chelates of tetramethyl-1,10-phenanthroline do not appear to be chemotherapeutically active against P388 ascites cells in the mouse.


Characteristics of rat aortic baroreceptors with nonmedullated afferent nerve fibers.

Nervous activity was recorded from nonmedullated afferent baroreceptor fibers in the left aortic nerve of normotensive rats. The aortic arch was perfused in vitro with oxygenated Krebs-Henseleit solution. Two different patterns of aortic C fiber discharge were found. One type (5 of 29 fibers) had a regular discharge at constant pressure whereas the other type had an irregular discharge. The differences between the two types were independent of the mean steady state rates of discharge. The average threshold pressure for the receptors with the regular discharge was 87 mm Hg (range, 65-125); it was 152 mm Hg (range, 85-260) for the irregularly discharging receptors. During a pressure step the receptors in either group normally showed an initial high frequency burst of activity and a sustained discharge which had a lower frequency. Furthermore, the peak discharge and the sustained discharge were lower on the average in the receptors with the irregular type of firing. We also found that addition of norepinephrine to the perfusate lowered threshold. Since unmyelinated axons are the predominant fiber type in the rat aortic nerve and since some receptors are activated at physiological pressures they may contribute to the tonic control of blood pressure. Their effects should be particularly important when the blood pressure is increased.


Intrarenal renin-angiotensin-sodium interdependent mechanism controlling postclamp renal artery pressure and renin release in the conscious dog with chronic one-kidney Goldblatt hypertension.

We studied postclamp renal artery pressure and renin release in eight conscious dogs with one-kidney Goldblatt hypertension. On normal sodium intake, intrarenal blockade of angiotensin II with Sar1-Ala8-angiotension II (P-113, saralasin acetate) markedly decreased postclamp renal artery pressure and increased renin release during the first 5 days after renal artery constriction. We found that 10-14 days after renal artery constriction, the maintenance of postclamp renal artery pressure and negative feedback on renin release became markedly less dependent on angiotensin II, as shown by almost no change in postclamp renal pressure or renin release with intrarenal blockade of angiotensin II. At this stage of our study the dogs were given a sodium diet of less than 5 mEq/day and we found that within 5-10 days intrarenal blockade of angiotensin II once again markedly decreased postclamp renal artery pressure and increased renin release. These observations support the concept of an angiotensin II-sodium interdependent negative feedback mechanism for renin release.


Pressor response to 1-sar-8-ala-angiotensin II (saralasin) in hypertensive subjects.

An angiotensin II (A II) analogue (1-Sar-8-Ala-angiotensin II) (saralasin) was infused into 418 untreated hypertensive subjects during a 1-day evaluation while blood pressure was recorded every 2 minutes by Arteriosonade. At 5 mug/kg per min, saralasin produced a change in mean blood pressure which correlated significantly (r=-0.54, P less than 0.001) with the stimulated plasma renin activity (PRA) (after intravenous furosemide and ambulation for 2 hours. Saralasin caused a rise inmean blood pressure of at least 7.0 mm Hg in 97 hypertensive subjects, who also had a low stimulated PRA (1.3+/-SEM, 0.1 ng/ml per hour; normal range, 1.7-8.5). On a low sodium diet, the pressor response of hypertensive subjects to saralasin continued and was an even better indicator of a low stimulated PRA. Infusion of saralasin at 10 mug/kg per min into normal subjects on an unrestricted diet, a low sodium diet, and a high sodium diet produced, respectively, no change, a fall (P less than 0.05), and a rise (P less than 0.005) in blood pressure. The same saralasin dose in six hypertensive subjects who showed a pressor response to the analogue in the 1-day study also produced a rise in blood pressure when given on a low sodium diet, and this rise was more than twice that seen in normal subjects on a high sodium diet. Hypertensive subjects who showed the pressor response had a significantly greater (P less than 0.01) pressor sensitivity to A II than did hypertensive nonresponders to saralasin and noraml subjects on an uncontrolled diet. The affinity of the vascular receptors for the analogue was greater in the hypertensive group that showed the pressor response to saralasin. In summary, the pressor response to saralasin, as defined above, occurred in 23% of a large unselected group of hypertensive subjects and was associated with salt loading, a low stimulated PRA, and increased pressor sensitivity to A II.


Length-dependent activation: its effect on the length-tension relation in cat ventricular muscle.

In cat papillary muscles at 30 degrees C, bathed with Tyrode's solution containing 2.25 mM Ca2+, the effect of various inotropic interventions (varying the stimulus frequency and continual paired stimulation) on the shape of the steady state length-tension relation was examined at lengths from Lmax, where tension production is maximal, to 0.87 Lmax. The relative steepness of the length-tension curves for peak tension developed (DT) and for maximum rate of tension development (dT/dT) varied inversely with the degree of potentiation. Thus, during paired pulse stimulation the relative decline in DT and dT/dT for a given change in muscle length was significantly less than the decline observed during stimulation at 5 min-1. When a muscle was stretched DT did not reach its final steady level for several minutes, and this slow increase in DT contributed significantly to the steepness of the steady state length-tension relation. The half-time of the slow increase in DT exhibited beat-dependency, and conditions that reduce the transsarcolemmal influx of calcium (reduction in bathing [Ca2+] or the presence of verapamil) significantly prolonged the time course of the slow increase and reduced its magnitude. These results support the hypothesis (1) that there is length-dependence of the excitation-contraction coupling process, such that an increase in muscle length is accompanied by greater activation of the contractile system; and (2) that this is due at least in part to an increased influx of calcium into the muscle cells. The implication of this hypothesis is that the influence of muscle length on myocardial performance (the Frank-Starling relation) should not be regarded as fundamentally different in character from other inotropic interventions.