Determination, by carbon-13 nuclear magnetic resonance spectroscopy, of the composition of glucans synthesized by enzymes of the cariogenic organism Streptococcus mutans. The 13C-n.m.r. spectra of water-soluble and -insoluble glucans synthesized by enzymes isolated from six strains of Streptococcus mutans are interpreted. The glucans are shown to be composed primarily of alpha-(1 linked to 3)- and alpha-(1 linked to 6)-linked glucosyl residues, and the relative abundance of each linkage is estimated from peak areas. Treatment of water-insoluble glucans with dextranase is found to result in water-soluble and -insoluble products, the former enriched in alpha-(1 linked to 6)-linkages and the latter in alpha-(1 linked to 3)-linkages. The structural conclusions arrived at by 13C-n.m.r. spectroscopy are consistent with data from methylation analysis and 1H-n.m.r. spectroscopy.


Stroke--1978.

Stroke--1978. The future of cerebral vascular disease management depends upon a sound biochemical and physiological understanding of the processes leading to stroke. As these become defined, new forms of therapy will arise with greater promise for treatment of the constellation of factors and symptoms known as stroke.


The characterization of human uterine smooth muscle cells in culture. Primary cultures initiated from normal human uterine endometrium after total enzymatic dissociation contained epithelioid cells and smooth muscle cells. The smooth muscle cells were subsequently isolated by differential trypsinization and grown in culture for 36 +/- 4 generations. Ultrastructural examination of log and post-confluent cultures of cells at low and high population doubling levels revealed characteristics similar to those of published reports on other smooth muscle cells studied in vivo and in vitro. Among the common features present were: (a) abundant bundles of 60--70 A myofilaments; (b) branched mitochondria; (c) stacks of cisternae of rough endoplasmic reticulum; (d) caveolae intracellulares; (e) nexuses. Other features included ovoid nuclei, a well developed Golgi apparatus and abundant free ribosomes. The subcultured cells exhibited features of dedifferentiation in the log phase of growth and at post-confluency. However, the post-confluent cells showed characteristics indicating redifferentiation back towards their in vivo morphology. Smooth muscle cells isolated from endometrial curettings may provide a useful model for biochemical and pharmacological studies of a cell type derived from a hormonal target tissue as the cells "age" in culture.


Phagocytosis of cells in the gastric surface epithelium of the rat. Gastric surface epithelial cells (SEC) from fed rats, from rats fasted for 16 h and from mucosae exposed in an ex-vivo chamber to 16 mM aspirin for 5 min were examined by transmission electron microscopy. SEC have the capability to phagocytose adjacent epithelial cells and parietal cells. Phagocytosis is rare in mucosae from fasted animals but common in fed animals or after brief exposure to aspirin. Phagocytic capabilities are not restricted to the progenitor zone but exist throughout the surface epithelium. Phagocytosis may provide a mechanism for the removal of damaged or senescent cells from the surface epithelium.


Evidence for actin transformation during the contraction-relaxation cycle of cytoplasmic actomyosin: cycle blockade by phalloidin injection. 1) The injection of a mushroom drug, Phalloidin (750 microgram -1 mg/ml), into the endoplasmic channel of Physarum veins induces an irreversible blockade of the intrinsic contraction-relaxation automaticity of the ectoplasmic tube wall, as measured by tensiometrical methods. 2) The morphological responses to Phalloidin injection include an increase and condensation of cytoplasmic actomyosin sheets bordering the plasmalemma invaginations within the ectoplasmic tube and a more pronounced dense layer of "groundplasm" in the cell cortex. This is in accordance with experiments with other cells as well as with Physarum. 3) The addition of marker particles to the injection solution revealed that the injected substances can be brought into direct contact with the contractile substrate, before newly formed membranes separate off the injection fluid. 4) Since Phalloidin irreversibly transforms oligomeric actin into a filamentous "Phalloidin-actin complex" and because this transformation does not hinder the actin in activating myosin ATPase, it is concluded that the contraction-relaxation cycle of cytoplasmic actomyosin in Physarum involves actin transformations. If these transformations are hindered, e.g. by Phalloidin, one stage and thereby the whole cycle is sustained which results in a blockade of the intrinsic contraction automaticity. 5) The functional importance of actin transformations in the congraction physiology of cytoplasmic actomyosins and cell motility phenomena is discussed.


The fine structure of the ocelli of Schistocerca gregaria. The neural organisation of the synaptic plexus. A study of the organisation of the locust dorsal ocellus shows that the structure is designed to provide the maximum possible effective aperture. The condenser-like cuticular lens and the dispersal of the rhabdome over a large proportion of the circumferential area of the retinula cells increases the light gathering power of the eye. The synaptic plexus of the ocellus has two major features: (i) the retinula cells are repeatedly and reciprocally connected by synapses and junctions, and (ii) there is an extensive lateral and feedback network between the receptors and interneurons. A unified structure is described for a synapse that presents differing profiles dependent upon the angle of section. A distinct morphological class of junction is described between retinula cells. The synaptic arrangements of morphologically identical retinula cells vary from cell to cell and the synaptic plexus is not organised with a high degree of spatial precision. The overall synaptic configurations are discussed in terms of the varied response characteristics of units in the ocellar nerve.


An ultrastructural stereological analysis of the aortic body chief cell of adult rabbits. The chief cells of the aortic body (subclavian body) of adult New Zealand white rabbits were examined by ultrastructural stereological analysis. The chief cell nuclei occupy 26.5% of the total volume. Dense-core vesicles account for 16.5% of the cytoplasmic volume, followed by mitochondria (11.6%), endoplasmic reticulum (3.3%), and Golgi apparatus (0.6%). The dense-core vesicles measure approximately 131.6 nm in diameter (corrected) and exhibit a heterogeneous size distribution. Both perivascular adrenergic nerve terminals and presumptive afferent terminals presynaptic to the chief cells are observed. The mean synaptic vesicle size of the terminals adjacent to chief cells is 54 nm. The heterogeneous size distribution of the dense-core vesicles of chief cells may indicate the storage of different biogenic amines and/or different secretion or maturation states within the chief cells.


Ultrastructure of the Y-organ of Astacus astacus (L.) (Crustacea) in relation to the moult cycle. The electron microscopical investigation of Y-organs of Astacus astacus revealed that during intermoult (stage C) the cytoplasm is poorly developed and that it increases at premoult (stage D). It then shows the typical signs of steroid production, namely agranular endoplasmic reticulum and mitochondria of the tubular type. Furthermore, a larger type of mitochondria with a regular pattern of internal structure is described.


The fiber composition of the semitendinosus muscle of the rabbit. The semitendinosus muscle of the rabbit is composed of a homogeneous fiber population. Fiber typing was carried out by means of light and electron microscopy according to which this muscle exhibits structural features that are either characteristic for both "red" and "white" fibers, or that do not allow for any classification according to the A-, B-, C-fiber system. Hence the long-held assumption that the semitendinosus muscle of the rabbit represents a classical paradigm of a "red" muscle should be revised in light of the present results.


Fixed phagocytes in the freshwater snail Lymnaea stagnalis. Morphological and histochemical examination of the blood and connective tissue of the freshwater snail Lymnaea stagnalis injected with various types of foreign particulate materials has shown the existence of free as well as fixed phagocytic cells. The morphology of the fixed phagocytes is described, and the phagocytic system of the snail is compared with that of other molluscan species.


Nucleus praeopticus and nucleus lateralis tuberis of Salmo salar and Salmo gairdneri: structure and relationship to the hypophysis. The nucleus praeopticus (NPO) is located on both sides of the preoptic recess and is composed of a pars parvocellularis and a pars magnocellularis. Only in the rainbow trout does the pars magnocellularis consist of separately located medium-sized cells and very large cells. Cytologically, three cell types can be distinguished: 1) unipolar cells ending in the cerebrospinal fluid (CSF), 2) bipolar cells also ending in the CSF and forming an axon, and 3) multipolar cells which generally do not have a direct connection with the ventricle. Axons originate from the cell bodies forming the paired preopticohypophysial tract that runs along the border of the diencephalon and the optic tract. A considerable number of NPO fibers leading to the hypophysis makes close contact with the cell bodies of the pars lateralis of the nucleus lateralis tuberis, indicating a functional relationship. Most NPO fibers terminate in the caudal part of the neurohypophysis, around blood capillaries and at the basal lamina of the pars intermedia. Far fewer fibers appear to terminate near the boundary of the neurohypophysis and the rostral and proximal pars distalis. The nucleus lateralis tuberis (NLT) is located in the caudal hypothalamus, beginning at the rostral end of the horizontal commissure and extending caudally beyond the hypophysial stalk. It consists of the partes rostralis, medialis, lateralis and ventrolateralis. In both species the p. rostralis contains small subependymal neurons and some larger ones. Only in the p. medialis of the Atlantic salmon are large cells present. In both species the most prominent part is the p. lateralis, which consists solely of large cells. Cells situated between the p. medialis and the p. lateralis are grouped in the p. ventrolateralis. It was impossible to trace the axons originating in the NLT, since the cyto- and axoplasm could not be stained specifically. The structure of the NPO and NLT in the two salmonid species is compared with that of other teleosts.


The fine structure of the vesicular component of the ultimobranchial gland of the domestic fowl. The ultrastructure of the polymorphic vesicular component of the ultimobranchial gland of the domestic fowl (Gallus gallus domesticus) has been described in detail, together with the structure of the cell strands interconnecting the vesicles and the parathyroid nodules lying within the ultimobranchial stroma. The vesicles frequently appear to arise from the nodules by way of the cell strands. The strands show a structure of their component cells intermediate between that of the parathyroid and the vesicular cells, although the position at which the strand changes from an essentially parathyroid structure to an essentially vesicular structure is very variable. The degree and kind of secretory activity within different cell types has been described. A review of the structure of ultimobranchial glands throughout the vertebrates shows that similar tissue with a similar secretory potential has been observed in all vertebrate classes, suggesting a functional significance for this part of the gland.


Ultrastructure of the subfornical organ of the chicken (Gallus domesticus). The SFO of the chicken is divided in half by a large central blood sinus; ventrally it is covered by a thin layer of ependyma (including tanycytes, dendrites, and axons) which connects the two lateral halves and protrudes as a  midsagittal crest into the lumen of the third ventricle. The ependyma consists predominantly of tanycytes with long basal processes which terminate upon perivascular spaces. These cells have an extensive Golgi apparatus and abundant lysosomes; their cellular apices containing polyribosomes and a few vesicles frequently protrude into the ventricle. In addition to astrocytes, oligodendrocytes, and microglial cells, there is another glial cell population that is distinguished by the presence of parallel stacks or spherical to ovoid conglomerates of rough ER and their unique location, i.e., limited to areas ventral and ventral-lateral to the large blood sinus. Two types of neurons are present: neurons in which there is a paucity of granulated vesicles and occasional vacuoles in both the cytoplasm and nuclei, the second type of neuron elaborates many granulated vesicles. Numerous puncta adhaerentia are observed between adjacent neuronal perikarya and between glial processes and neuronal perikarya. Diverse axon types are found within the chicken SFO. Axo-dendritic and axo-somatic axon terminals and presynaptic axon dilations contain assorted combinations of electron-lucent and granulated vesicles of different maximal diameters. Based on the morphology of these axons, cholinergic, peptidergic, and serotoninergic fibers are described. There are two additional groups of axons whose classification awaits further investigation. The chicken SFO differs from the mammalian SFO in several respects: it possesses an ependyma with secretory and/or absorptive tanycytes predominating; it is divided midsagittally by a central blood sinus; its lateral and dorsal limits are nebulous; a previously undescribed peculiar type of glial cell is found in a limited portion of the organ; supraependymal neurons are lacking.


Fluorescent histochemistry of the teleost gut: evidence for the presence of serotonergic neurones. The guts of the teleosts Myoxocephalus scorpius, Pleuronectes platessa and Clupea harengus were examined using fluorescent histochemistry and the innervation compared to that in mammals and other lower vertebrates. In Myoxocephalus and Pleuronectes both greeen and yellow fast fading fluorescent nerves were found in the intestine. To test whether the yellow nerves might be serotonergic, an attempt was made to extract 5-HT from the intestine of these fish in a region where enterochromaffin cells were absent. Chromatography of extracts showed the presence of 5-HT, and, in Myoxocephalus, dopamine.


Early detection of inherited muscular dystrophy in chickens. New Hampshire chickens, homozygous for inherited muscular dystrophy, display clinical manifestations at an early age. A fine structural examination of embryos from this strain shows marked degenerative changes four days prior to hatching. The Z bands appear to dissolve progressively to the point where finally the myofibrils become uniformly dense with no detectable banding patterns.


Scanning electron microscopic analysis of the linings of the fourth ventricle in the domestic fowl. Surface features of the ependymal linings of the fourth ventricle in the fowl were analyzed employing the scanning electron microscope (SEM). On the floor of the median sulcus, each ependymal cell has a solitary cilium, whereas on both sides of the sulcus, cilia are so densely distributed that the details of the underlying cell surface are usually obscured. On the roof of the fourth ventricle, except for the surface of the ciliated groove where numerous cilia are present, the ependymal cells are polygonal in shape, and the center of each cell possesses an aggregate of ten to twenty cilia. Cell surfaces of the choroid tela are entirely covered with delicate microvilli and possess clumped cilia. The ependymal cell surfaces of the area postrema are dome-like in shape. Each ependymal cell has a solitary cilium and shows a smooth surface free of microvilli.


The organization of the lamina ganglionaris of the hemipteran insects, Notonecta glauca, Corixa punctata and Gerris lacustris. Neuronal elements, i.e. first and second order neurons, of the first optic ganglion of three waterbugs, N. glauca, C. punctata and G. lacustris, are analyzed on the basis of light- and electron microscopy. Eight retinula cell axons, leaving each ommatidium, disperse to different cartridges as they enter the laminar outer plexiform layer. Such a pattern of divergence is one of the conditions for neuronal superposition; it is observed for all three species of waterbugs. The manner in which the receptors of a single bundle of ommatidia split of within the lamina, whereby information from receptors up to three or five horizontal rows away can converge upon the same cartridge, differs among the species. Six of the eight axons of retinula cells R1--6, the short visual fibers end at different levels within the bilayered lamina, whereas the central pair of retinula cells R7/8, the long visual fibers, run directly through the lamina to a corresponding unit of the medulla. Four types of monopolar cells L1-L4 are classified; their branching patterns seem to be correlated to the splitting and termination of retinula cell axons. The topographical relationship and synaptic organization between retinula cell terminals and monopolar cells in the two laminar layers are identified by examination of serial ultrathin sections of single Golgi-stained neurons. An attempt is made to correlate some anatomical findings, especially the neuronal superposition, to results from physiological investigations on the hemipteran retina.


Unusual granules in the ejaculatory duct of a Microphthalmus species (Polychaeta, Annelida). The paired prominent ejaculatory ducts of the hermaphroditic polychaete Microphthalmus cf. listensis are surrounded by gland cells the processes of which penetrate the ducts themselves. These cells produce, in separate regions, two different types of spherical granules. Type I is composed of an electron dense and an electron lucent part. Type II granules contain a tubular filament that forms a single or double spiral in the periphery of a more or less unstructured electron dense material. Golgi vesicles give rise to this granule type. During the passage of sperm, these granules are obviously discharged into the lumen of the duct. Here they change form and probably dissolve. Their function is as yet unknown; capacitation of sperm is assumed.


Lysosomal breakdown of erythrocytes in the sheep placenta. An ultrastructural study. The breakdown of erythrocytes within the lysosomal apparatus of trophoblastic epithelial cells of the sheep placenta was studied at the ultrastructural level. Acid phosphatase activity could be demonstrated in the interspace between the erythrocyte membrane and the lysosomal membrane, but not inside ingested erythrocytes. The erythrocyte plasma membrane remained observable until the final stage of the breakdown process. Together with a peripheral layer of indigestible hemoglobin it might form a barrier for further penetration of lysosomal enzymes into the ingested erythrocyte. The hemoglobin of the erythrocyte is suggested to diffuse through the erythrocyte plasma membrane into the interspace between this membrane and the lysosomal membrane. Subsequently, the hemoglobin is digested in the interspace or in fragments pinched off from erythrocyte-containing lysosomes (= erythrolysosomes). The fragmentation of erythrolysosomes is considered to be the most efficient mechanism for the breakdown of red blood cells in the trophoblastic epithelium of the sheep placenta. The method of entry of hydrolytic enzymes into erythrocyte-containing phagosomes is discussed.


The differentiation of monocytes into macrophages, epithelioid cells, and multinucleated giant cells in subcutaneous granulomas. I. Fine structure. The morphological changes occurring in monocytes during their differentiation into macrophages, epithelioid cells, Langhans-type giant cells, and foreign-body-type giant cells were investigated in foreign-body granulomas induced by subcutaneous implantation of pieces of Melinex plastic. Analysis based on Adams's (1974) criteria for discrimination between the several types of cell of the monocyte line, showed that each type has a characteristic type of granule. Primary and secondary granules, numerous in the Golgi area of monocytes were generally found close to the cell membrane and decreased in number in maturing macrophages. This was accompanied by an increase in the number of microtubules. Mature macrophages show numerous characteristic macrophage granules, which are round (average diameter: 280 nm) and have a halo between the limiting membrane and granular matrix. Mature epithelioid cells have characteristic epithelioid cell granules, and multinucleated giant cells a heterogenous population of granules. Fusing macrophages generally have their Golgi areas facing each other, and also show a reduced thickness of the cell coat. The morphology of the multinucleated giant cell is closely related to the number of nuclei present. In Langhans-type giant cells, which generally have two to ten nuclei, a giant centrosphere with numerous aggregated centrioles is found. In transition forms between Langhans-type and foreign-body-type giant cells, which generally contain 10--30 nuclei, the centrioles show less aggregation. In the foreign-body-type giant cells, which generally have more than 30 nuclei, centrioles are virtually absent and never aggregated. These differences between the Langhans-type giant cells, the foreign-body-type giant cells, and the transition forms, support our previous finding that Langhans-type giant cells are the precursors of foreign-body-type giant cells.


The differentiation of monocytes into macrophages, epithelioid cells, and multinucleated giant cells in subcutaneous granulomas. II. Peroxidatic activity. The peroxidatic (PO) activity of monocytes differentiating into macrophages, epithelioid cells, and multinucleated giant cells in subcutaneous granulomas was investigated with three different media for the demonstration of PO activity. Irrespective of the stage of differentiation, these cells did not show PO activity in the rough endoplasmic reticulum (RER) or nuclear envelope. In addition, it was found that the morphologically characteristic types of granule of the various cells of the monocyte line (the primary granules and secondary granules of monocytes, the macrophage granules, and the epithelioid cell granules), all have distinct cytochemical characteristics. Monocytes lose their primary and secondary granules during differentiation into mature macrophages. Simultaneously, the granules of both types become elongated and the secondary granule lose their halo. In contrast to monocytes, mature macrophages may contain a few microperoxisomes. During the differentiation of macrophages into epithelioid cells or multinucleated giant cells there is an increase in the number of microperoxisomes.


Comparative study on peroxidatic activity in inflammatory cells on cutaneous and peritoneal implants. Inflammatory reactions were evoked by simultaneous implantation of pieces of Melinex plastic in the subcutaneous tissue of the dorsum and in the peritoneal cavity of rats. The cellular composition of the Melinex-adherent cells and their peroxidatic (PO) activity were investigated in relation to the duration of implantation. Several striking differences were found between the subcutaneous and peritoneal implants. On the 7th and 14th days, multinucleated giant cells were abundantly present on the subcutaneous implants, whereas they were relatively rare on the peritoneal implants. The subcutaneous implants bore no mast cells and only a few eosinophilic granulocytes, but both types of cell were observed frequently on the peritoneal implants. Macrophages and multinucleated giant cells on the subcutaneous implants show PO activity only in the granules are PO negative. On the peritoneal implants three types of macrophages can be dintinguished: exudate macrophages which have PO activity restricted to granules or are PO-negative; macrophages with PO activity in granules and both the rough endoplasmic reticulum (RER) and nuclear envelope; and resident macrophages with PO activity only in the RER and nuclear envelope. In addition, two types of multinucleated giant cells are found, one with and the other without PO activity in the RER and nuclear envelope. Multinucleated giant cells with PO activity in the RER and nuclear envelope as well as exudate macrophages with PO activity in the RER and nuclear envelope were mainly found 32 h and 3 days after implantation of the Melinex in the peritoneal cavity. These findings are discussed in the light of current knowledge of the PO activity in macrophages and multinucleated giant cells. It is concluded that the appearance of PO activity in the RER and nuclear envelope of exudate macrophages and multinucleated giant cells is in all probability a transient phenomenon, and that there is no objective evidence to support the opinion that exudate macrophages with PO activity in the RER and nuclear envelope are transitional cells between exudate and resident macrophages.


A correlated scanning and transmission electron microscopic study of maturation ameloblasts in developing molar teeth of rats. Maturation ameloblasts of developing molar teeth of the rate were studied by both scanning and transmission electron microscopy. After fixation, teeth were frozen and split. One face of the fractured tooth was used for SEM, the other for TEM. It was found that in some regions proximal junctional complexes separate the interameloblast space from the intercellular space of the papillary layer. Thereby an intercellular ameloblastic compartment is delineated which in some specimens contains a substance interpreted to be colloidal. Elsewhere the proximal junctions of ameloblasts are not present and free communication between the extracellular spaces is evident. The apical pole of ameloblasts varies in structure. Over some areas there is a distinct distal border zone with membranous infoldings which in some regions resembles a striated or ruffled border, but in other regions the membranes show whorl configurations. The distal border zone also contains granules with flocculent material. Elsewhere the ameloblasts display no distal border zone and the cells show a smooth membrane (except for pinocytotic vesicles and hemidesmosomes) facing the enamel surface. The lateral surface of ameloblasts exhibits a variety of surface configurations similar to but not as pronounced as those reported previously in rat incisor maturation ameloblasts.


Freeze-fracture study of the chromaffin cell during exocytosis: evidence for connections between the plasma membrane and secretory granules and for movements of plasma membrane-associated particles. Exocytosis was studied in acetylcholine-stimulated bovine adrenal medulla. During a pre-exocytotic stage, chromaffin granules are found in juxtaposition to the plasma membrane and separated from it by an electron dense space 25--27 A in width. Freeze-fracture studies show this stage to be characterized by connections between the granules and the plasma membrane. These connections are apparently cytoplasmic but bridge both membranes; they are presumably proteinaceous, but their exact nature remains to be elucidated. Later stages of exocytosis were also studied by the freeze-fracture technique; a typical feature is the lack of intramembrane particles around the fusion site. Both connections and membrane particle movement are discussed in terms of recent biochemical findings.


Physiological cell death of secretory ameloblasts in the rat incisor. The migration of the ameloblasts in the continuously erupting incisors of the rat is accompanied by cell loss. Ameloblasts degenerate near the mesial and lateral cemento-enamel junctions in the secretory zone and in the middle two thirds of the region of postsecretory transition, degeneration being most marked where these areas merge. These findings support the hypothesis that the prism decussation in the enamel results from alternating transverse rows of secretory ameloblasts sliding past each other whilst elaborating their rods. The distribution of the degenerating cells suggests, however, that the sliding cell rows are not exactly transverse but arcuate, with the opening facing incisally. The progress of structural alterations of the nuclei in the degenerating ameloblasts appears to follow the pattern earlier described in vinblastine-damaged ameloblasts.


Effects of caffeine and D2O on persistence and de novo generation of intrinsic oscillatory contraction automaticity in Physarum. The present investigation was performed in an attempt to contribute to answering the question whether the plasmalemma of the plasmodial stage of Physarum represents the site of a trigger mechanism for the oscillating contraction activity of cytoplasmic actomyosin. The effects of the following substances on persistence of tensiometrically measured longitudinal and radial activities of Physarum veins and on de novo generation of activities in experimentally generated drops were studied: caffeine, theophylline, acetylcholinium chloride, procaine, physostigminium salicylate, iso-ompa, nifedipin, sodium nitroprusside, potassium thiocyanate, D2O; as well as the effects of ions such as La+++ and high outer concentrations of Na+ and K+. Some of the substances were applied simultaneously for comparison externally (by bathing solutions) and internally (by injection). The experimental data speak against the existence of electrogenic rhythmical Ca++, Na+ or K+ pumps across the plasmalemma which could have a triggering function for the oscillation. The contraction activities of the cytoplasmic actomyosin seem to represent a spontaneous endogeneous oscillation which can be modulated via the plasmalemma during chemotaxis.


The juxtaligamental cells of Ophiocomina nigra (Abildgaard) (Echinodermata: Ophiuroidea) and their possible role in mechano-effector function of collagenous tissue. The intervertebral ligament of the brittlestar Ophiocomina nigra contains numerous cellular processes which belong to perikarya located on the outer surfaces of the ligament. These are described as the juxtaligamental cells and have been studied by light and electron microscopy. The cells are mainly concentrated in four pairs of ganglion-like nodes associated with the intervertebral ligament and in similar nodes adjacent to every other major connective tissue component of the arm. Although their histochemistry and ultrastructure indicate a neurosecretory function, they are anomalous in containing unusually large electron-dense granules probably associated with calcium. The ganglion-like nodes are innervated by hyponeural nerves, though synaptic contacts with the juxtaligamental cells have yet to be demonstrated. The function of the cells is discussed and it is suggested that they may be involved in the rapid loss of tensil strength which the intervertebral ligament sustains during arm autotomy. They may achieve this by controlling the availability of Ca2+ ions to the extracellular compartment of the ligament.


Autoradiographic studies of a glucocorticoid agonist and antagonist: localization of 3H-corticosterone and 3H-cortexolone in mouse brain. The localization in the mouse brain of corticosterone, the natural glucocorticoid in the mouse, and cortexolone, reported to be a glucocorticoid antagonist, was studied by autoradiography 30 min after in vivo administration of the tritiated compounds. After 3H-corticosterone (3HB) injection, radioactivity was preferentially concentrated in cell nuclei of several structures within the limbic system, and in nuclei of certain neurones of the cerebral cortex and medullar oblongata. This nuclear concentration was abolished after injection of 3H-corticosterone with an excess of unlabelled corticosterone. After 3H-cortexolone (3HS) injection, a diffuse radioactivity was observed throughout the brain. However, a higher concentration of grains was present in the ventral nucleus arcuatus and in the infundibulum. When excess unlabelled cortexolone was administered with 3H-cortexolone this preferential accumulation of grains was abolished. The accumulation of 3H-cortexolone in the medial basal hypothalamic region suggests that cortexolone concentrates preferentially in dexamethasone (DM) target regions, and in addition the autoradiographic results show that the cortexolone-receptor complex does not accumulate in the cell nucleus.


Scanning electron microscopy of the ventricular surface of the paraventricular organ in the domestic fowl. The ventricular surface of the paraventricular organ in the domestic fowl is covered with supraependymal fiber elements. These fibers from a dense network having a web-like appearance and a coarse network of fibers running over the ventricular surface. The dense, web-like networks are mainly distributed throughout the caudal region of this organ, whereas the course networks are observed in the rostral region. In addition to these structures, spherical bodies with diameters approximately 2.5 to 6 micron are also encountered.


The sensitivity of contrast echocardiography in detecting intracardiac shunts. While contrast echocardiography has been shown to delineate intracardiac shunts, no information is available concerning the sensitivity of the method. This study establishes the relative sensitivity of the technique in detecting and localizing shunts by comparing the contrast echocardiograms with the simultaneously recorded cardiogreen dye curves in 26 children during cardiac catheterization. The echocardiograms substantiated isolated intracardiac shunting as small as 5%. There were no false-positive or false-negative echocardiograms in patients with or without shunt defects. This report demonstrates the contrast echocardiographic technique to be extremely sensitive in detecting intracardiac communications. It reviews the diagnostic patterns necessary for diagnosis and proposes the technique as a sensitive, safe, and accurate alternate method of study in the cardiac catheterization laboratory.


The differential diagnosis of paracardiac lesions: pericardial cysts. The differential diagnosis of paracardiac lesions includes pericardial cysts (PC), which are benign, developmental lesions. Patients with PC are usually asymptomatic, although chest pain or dyspnea may occur. The diagnosis may be established by chest roentgenogram (radiographic contour and location), fluoroscopy (changes in shape with respiration or positioning the patient), and echography (smooth, cystic contour and characteristic location). We present the diagnostic approach to patients with PC, and a review of the case histories of 12 patients with PC. Two symptomatic patients with PC were treated with surgical excision. Cyst aspiration in two patients yielded clear fluid and was initially considered therapeutic, but was followed by gradual reaccumulation of fluid. Six of ten asymptomatic patients followed for three to ten years did not develop symptoms, nor was there radiographic evidence of progressive PC enlargement. Three of ten died from unrelated causes during the follow-up period. Although symptomatic patients with PC may require surgical excision, asymptomatic patients with PC should be managed conservatively.


Diagnosis of left atrial tumors by coronary angiography and left ventriculography. The left ventriculograms and pulmonary and coronary angiograms of eight patients with left atrial (LA) tumors were reviewed. The patients' ages ranged from 52 to 65 years. In each case the tumor was outlined during left ventriculography. Coronary arteriography allowed visualization of tumor vessels in six cases (six of seven myxomas). Two of the patients had significant coronary obstructions. It is concluded that a left ventriculogram is a sensitive diagnostic test for the presence of LA tumors. Coronary arteriography is indicated in patients with LA tumors in this age group and is likely to provide independent evidence of the presence of an LA tumor.


Iliac venous thrombosis in infants and children after cardiac catheterization. Twenty-two patients developed thrombosis of the lower abdominal portion of the inferior vena cava, iliac or femoral vein (IVT) after cardiac catheterization. All patients has at least one previous study from 1 day to 11 months of age (mean, 2 months) and seven had two catheterizations before discovery of IVT. Transposition of the great arteries was the most common defect associated with IVT (12/22, 55%). From one to seven catheter changes were made during the time of venous cannulation (mean, 105 minutes). No clinical evidence of IVT was present immediately after the preceding cardiac catheterization. The discovery of significant IVT usually necessitated the use of a vein from the upper extremity to complete the cardiac catheterization. IVT was associated with previous balloon atrial septostomy using either a Fogarty or Rashkind septostomy catheter in 13/22 patients (59%). In addition, 86% of the patients had either of these balloon catheters or a Swan-Ganz catheter used during a previous study. The potential development of IVT should be considered especially in cyanotic infants and small children in whom balloon catheters are used.


Tricuspid regurgitation: clinical and angiographic assessment. Because previous attempts to diagnose and quantitate tricuspid regurgitation (TR) by angiography have been unreliable, 60 patients with mitral or combined mitral and aortic valve disease had right ventriculography using a special preshaped catheter. A clinical diagnosis of TR was confirmed in 45% of the patients with moderate and severe TR. A pansystolic murmur increasing in intensity with inspiration, a pulsatile liver, and a prominent CV wave in the jugular veins when present together were specific for severe TR but were seen in only 30% of the patients with severe TR but were seen in only 30% of the patients with severe TR (3+ or 4+). Ventricularization of the right atrial pressure contour was specific for severe TR but was seen in only 40% of the patients with severe TR. A normal right atrial mean pressure (RAP) did not exclude TR, but a rise in RAP or an unchanged RAP with deep inspiration was seen in all patients with TR. Similar findings were observed in two patients with severe pulmonary hypertension who had no TR. There was no relation between the magnitude of this rise in RAP, the degree of pulmonary hypertension, and the severity of TR. The use of a special preshaped catheter tends to avoid the induction of premature beats, and right ventriculograms with a preshaped catheter may be useful in diagnosing TR.


Cor triatriatum with mitral regurgitation in a 52-year-old male. The clinical, echocardiographic, and cineangiographic features of the oldest known living case of cor triatriatum are presented. Mitral regurgitation, also found in this patient, has been reported on only two previous occasions to coexist with other wise uncomplicated cor triatriatum. The value of two-dimensional echocardiography in the identification of this congenital anomaly is confirmed.


The noninvasive assessment of anomalous origin of the left coronary artery from the pulmonary artery. We present a patient with anomalous origin of the left main coronary artery from the pulmonary artery. We correlate the findings of echocardiography and myocardial imaging with angiography, and discuss the value of the noninvasive techniques in the diagnosis and in the followup of such patients.


A simple, new system for maintaining measured quantities of saline cold and sterile for thermodilution cardiac output measurement. A new, simple system for cooling and maintaining prefilled syringes of injectate in a dry, sterile environment for thermodilution cardiac output using a thermoelectric cold plate is described. Rates and uniformity of cooling were studied. Uniform cooling is achieved in about 60 minutes if refrigerated injectate is used.


Multiple sites for the initiation of microtubule assembly in mammalian cells. The pattern of microtubule regrowth in mammalian fibroblast and epithelial cells has been examined by immunofluorescence of cytoskeletal preparations with antibody to tubulin. After reversal of treatment with colcemid, vinblastine or low temperature, microtubules appear to grow simultaneously from several distinct initiation sites located within 5 microns of the nucleus of mouse and human fibroblasts. Each site initiates the growth of 10-30 microtubules. More than 70% of the mouse fibroblasts have between 5 and 10 initiation sites with an average of 8. The human fibroblasts have an average of 5 sites per cell. The average number and numerical distribution of sites per fibroblast cell are not affected by time of exposure to colcemid or the concentration of colcemid applied to the cells. Multiple microtubule initiation sites are also observed during the process of microtubule depolymerization. In addition to growth from these complex initiation sites, microtubules appear to grow singly from the perinuclear region of human fibroblasts. The regrowth of individual microtubules from the perinuclear growth is especially prominent in epithelial cell lines from rat kangaroo and pig. These epithelial lines have only a single complex initiation site per cell. Two classes of complex initiation sites can be distinguished in microtubule regrowth experiments in human and mouse fibroblasts after exposure to griseofulvin. Microtubules first grow extensively from a single distinct site, which has approximately 20 microtubules growing from it and may be the centriole or centriolar pair. Subsequently, microtubules regrow from other perinuclear complex initiation sites. It thus appears that at least three distinct classes of initiation sites can be observed in mammalian cells: primary sites, which regrow microtubules first after griseofulvin treatment; secondary sites, which are distinct perinuclear sites and recover from griseofulvin treatment more slowly than the primary sites; and tertiary sites or sites of growth of single microtubules, also located near the cell nucleus.


Aggregation of microtubule initiation sites preceding neurite outgrowth in mouse neuroblastoma cells. By examining microtubule regrowth using immunofluorescence with antibody to tubulin, we have studied the structure and intracellular localization of microtubule initiation sites in undifferentiated and differentiated mouse neuroblastoma cells. The undifferentiated cells are round and lack cell processes. They contain an average of 12 initiation sites per cell. Each of these sites, which are located near the cell nucleus, initiates the growth of several microtubules in a radial formation. In contrast to the undifferentiated cells, neuroblastoma cells stimulated to differentiate by serum deprivation are asymmetrical, containing one or two very long neurites. These cells have a single, large microtubule initiation center which can be visualized not only by immunofluorescence but by phase-contrast and differential interference microscopy as well. The initiation site measures 3-4 mu in diameter and is located in the cell body along a line defined by the neurite. During cell differentiation, the large initiation, the large initiation center seems to be formed by the aggregation of many smaller sites. This process procedes neurite extension by about 24 hr. The growth of microtubules from this center appears to be highly oriented, since most microtubules initially grow into the neurite processes rather than into the cell interior. Thus major changes in the structure and location of microtubule initiation sites occur during the differentiation of neuroblastoma cells. Similar changes are likely to be involved in alterations in the morphology of other cell types.


Binding of soluble type I collagen molecules to the fibroblast plasma membrane. Soluble 125I-labeled type I collagen binds to cultured fibroblasts but not to cultured epithelia. The binding of the ligand to fibroblasts is reversible, saturable and highly specific for sequences contained within the helical portions of the alpha1 and alpha2 chains. The amount of ligand bound is dependent upon cell number and ligand concentration. Binding is decreased but measurable at 4 degrees C. The steady state binding is greater at 26 degrees than at 37 degrees C due to a more rapid dissociation of the ligand-acceptor complex at 37 degrees C. The half-life of the complex is 46 min at 37 degrees C and approximately 2.5 hr at 26 degrees C. Scatchard plots of binding data indicate a single class of high affinity binding sites (KD = 1.2 X 10(-11) M) with each fibroblast binding approximately 500,000 molecules at saturation. Pretreatment of fibroblasts with bacterial collagenase, chondroitinase ABC or testicular hyaluronidase does not affect the binding reaction, whereas pretreatment of the cells with phospholipase C increases the amount of ligand bound. Ligand binding is decreased but not abolished after fibroblasts are treated with trypsin concentrations which remove surface fibronectin. Fibroblast monolayers treated with antiserum against fibronectin bind the radiolabeled ligand normally. In contrast to collagen, addition of excess fibronectin does not accelerate the dissociation of bound ligand from fibroblasts. Possible functions for surface-bound collagen are discussed.


Restriction endonuclease cleavage of linear and closed circular murine leukemia viral DNAs: discovery of a smaller circular form. Both linear (form III) and closed circular (form I) viral DNAs obtained from mouse cells infected with Moloney murine leukemia virus were cleaved by Sal I, Sma I, Bam HI and Pst I restriction endonucleases. DNA fragments generated by these cleavages were ordered with respect to the 5' and 3' ends of the RNA genome by several techniques, including comparisons of the DNA fragments from cleavages of the linear and closed circular forms, double digestions using different combinations of enzymes and the use of an RNA probe specific for the 3' end. DNA from Hirt extractions of infected cells yielded a discrete species of linear viral DNA whose size was determined by agarose gel electrophoresis to be 5.7 x 10(6) daltons. In the course of characterizing the closed circular DNA, we observed two form I DNA molecules. The larger molecule was the same size as the linear DNA. The second molecule migrated faster on agarose gels and was the predominant species of the two closed circular DNAs. Using the restriction endonuclease maps which we derived, we demonstrate that this novel form I DNA is a smaller homogeneous species of viral DNA, missing about 600 nucleotides found in the linear and larger closed circular DNA molecules. We have localized the site of this missing DNA piece to be at either one or both ends of the linear viral DNA.


Structural analysis of the fibroin gene at the 5' end and its surrounding regions. Using the genomic clones of the fibroin gene with its flanking sequences, a detailed restriction map was prepared with particular attention given to the region neighboring the 5' end of the gene. About 60% of the fibroin mRNA has a complete cap (m7GpppAmUmCXG), and the remaining 40% has an unmethylated cap (GpppAPyXXG). The latter can be radioactively labeled by the use of vaccinia capping enzymes. Using the labeled mRNA, we mapped the 5' end of the gene by hybridization to restriction enzyme digests. DNA sequencing of this region revealed a sequence ATCAGCATCAG that corresponds to ApyXXG in tandem. The 5' end of mRNA with a complete cap was also located in these 11 nucleotides. Since the second ATCAG region was protected from S1 digestion following hybridization with total fibroin mRNA, we have tentatively assigned it as the sole 5' end of the transcription unit. About 700 bp were sequenced in the region from -800 + 1700 bp. The region of about 210 bp preceding the 5' end of the gene which may accommodate a promoter sequence is very AT-rich and contains clusters of A and T residues. This region contains statistically significant dyad symmetries, direct repeats and inverted repeats. At -30 to -24, the sequence TATAAAA was observed, and this is similar to a Pribnow-type sequence (TATAGATG). The sequence that codes for the gly-ala repetitious peptide characteristic of fibroin begins somewhere between 1340 and 1600 bp from the 5' end. An intervening sequence of about 1.1 kb that interrupts the gene about 70 bp from the 5' end was detected by R loop formation. Structures of the fibroin gene cloned from fibroin producing and nonproducing cells look the same, especially around the 5' end and the intervening region.


Structure of DNA within three isometric bacteriophages. This paper describes a model for the structure of DNA contained in three morphologically similar bacteriophages--T7, P22 and phiCd-1--based on the transient electric dichroism of intact phage. The reduced dichroism of each of the phages at perfect orientation is within the range +0.12 to +0.19. Assuming that the phage orientation axis is that which passes from the apex through the tail, the measured dichroism suggests that DNA is wrapped in closely packed, co-axial solenoids with the axis of the solenoids tipped 43.5 degrees +/- 2.5 degrees from the orientation axis of the phage. All three phages show a large permanent dipole moment, with respective values of 5600, 200,000 and 500,000 Debye for T7, phiCd-1 and P22. The radius of the equivalent sphere for the three phages calculated from the rotational relaxation time for the rise of dichroism is in agreement with birefringence and electron microscope observations. The circular dichroism spectra of all three bacteriophages indicate that the local DNA helicity is similar in each case.


Vitellogenin in Xenopus laevis is encoded in a small family of genes. Vitellogenin, the yolk protein precursor, is produced in X. laevis liver from a 6.3 kilobase (kb) mRNA. Sequences of this mRNA have been transcribed into cDNA and cloned in E. coli. Some properties of 21 of these cloned DNAs, ranging in size from 1 to 3.7 kb, have been reported by Wahli et al. (1978b). This paper reports restriction endonuclease mapping, cross hybridization, heteroduplex mapping in the electron microscope and heteroduplex melting experiments with these DNAs. We conclude that the cloned DNAs fall into two main groups of sequences which differ from each other in approximately 20% of their nucleotides. Each main group contains two subgroups which differ from each other by about 5% sequence divergence. By hybridizing cloned DNAs with restricted genomic DNA, we showed that sequences corresponding to all four sequence groups are present in a single animal. Furthermore, we have obtained tentative evidence for the presence of large intervening sequences in genomic vitellogenin DNA. Analysis of R loop molecules demonstrated that all four sequences are present in the vitellogenin mRNA population purified from individual animals. While some alternate explanations are not entirely excluded, we suggest that vitellogenin is encoded by a small family of related genes in Xenopus.


Deletion mutants of Xenopus laevis 5S ribosomal DNA. Deletion mutants have been derived from a plasmid-cloned repeating unit of Xenopus laevis oocyte 5S DNA by introducing the transposable chloramphenicol-resistance element Tn9 into the AT-rich spacer sequence near the 5' terminus of the X. laevis 5S rRNA gene in a recombinant plasmid and then selecting plasmids which had lost the transposable element. Plasmids lacking the entire transposable element and various portions of the AT-rich spacer sequence flanking the original site of Tn9 integration have been obtained, and their ability to support transcription of the remaining X. laevis 5S rRNA gene has been tested in X. laevis oocyte nuclei. The deletion mutants analyzed in the present study retain the 49 nucleotide nonrepetitive sequence immediately adjacent to the 5' terminus of the gene, but lack as much as 80% of the repetitive AT-rich spacer sequence (Fedoroff and Brown, 1978). Such deletion mutants are fully active templates for 5S rRNA synthesis. This implies that the AT-rich spacer, which comprises half or more of each repeating unit in X. laevis oocyte 5S DNA, is relatively unimportant for correct initiation of transcription, and that if there are extragenic sequences with promoter function, they are likely to reside in the short nonrepetitive region immediately adjacent to the gene.


An intervening sequence in the gene coding for 25S ribosomal RNA of Tetrahymena pigmentosa. An intervening sequence of approximately 400 bp has been discovered in the 25S ribosomal RNA gene (rDNA) from macronuclei of Tetrahymena pigmentosa strain 6UM. Another strain, 8ALP, lacks the intervening sequence in its macronuclear rDNA. The presence of the intervening sequence was first suggested by a comparison of the restriction endonuclease maps of the rDNA from each strain. A region which differed in size between the two strains was shown to lie within the region coding for 25S ribosomal RNA by hybridization of the restriction fragments to labeled 25S rRNA. Direct visual evidence for the existence of the intervening sequence in 6UM and its absence from 8ALP was obtained by electron microscopy of R loop hybrids formed between the rDNA and 25S rRNA. The size and position of the intervening sequence within the gene were further defined by detailed restriction analyses of cloned fragments of the rDNA from each strain. The rDNA within the macronucleus of each of these strains is homogeneous, and thus the intervening sequence in the rDNA of strain 6UM must not interfere with transcription of the gene. Furthermore, the presence or absence of the intervening sequence has no obvious functional importance, since both strains can survive and grow equally well.


Acetylcholine receptor degradation measured by density labeling: effects of cholinergic ligands and evidence against recycling. The methodology of density labeling of proteins by biosynthetic incorporation of 2H, 13C, 15N-amino acids into newly synthesized polypeptide chains allows the direct measurement of the turnover rate of the acetylcholine receptor in cultured chick skeletal muscle. In this study, receptors synthesized in medium containing 2H, 13C, 15N-amino acids were resolved from 1H, 12C, 14N-receptors by velocity sedimentation in sucrose-deuterium oxide gradients, and their proportions were determined by computer analysis of the gradient profiles. The kinetics of turnover of acetylcholine receptors are identical for developing chick muscle fibers grown in medium containing 2H, 13C, 15N-amino acids or 1H, 12C, 14N-amino acids, and the high degree of substitution of normal aminoacyl residues by 2H, 13C, 15N-residues does not affect the turnover rate of the denser receptor. Comparison of the turnover rates in continuous and pulse-labeling experiments gave independent confirmation of these results. The application of a potent, essentially irreversible blocking agent, alpha-bungarotoxin, increases the median lifetime of receptors from 17 hr for the native unbound receptor to 22 hr for the alpha-bungarotoxin-receptor complex. As predicted, the total number of alpha-bungarotoxin binding sites increased in the continued presence of alpha-bungarotoxin due to extension of receptor lifetime. To determine whether other cholinergic agents affect the turnover rate of the receptor, measurements were performed on cultures grown in the presence of 10(-4) M d-tubocurare or 10(-4) M carbachol, a reversible antagonist and a reversible agonist, respectively, of the nicotinic acetylcholine receptor. The receptor degradation rates of the drug-treated cells were identical to control values. The total number of alpha-bungarotoxin binding sites was reduced by 30% in the presence of carbachol, indicating that this agent affects the rate of synthesis of the acetylcholine receptor. Data formerly interpreted as suggesting a cycling of receptor-containing plasma membrane out of and back into the sarcolemma are now understood to reflect the alteration in receptor lifetime upon complexing with alpha-bungarotoxin. The intracellular "hidden" receptor sites were found to remain inside the myotubes and thus do not signify an intracellular pool of recycling plasma membrane.


Physical structures of Tn10-promoted deletions and inversions: role of 1400 bp inverted repetitions. We report here the physical structures of deletions and inversions promoted by the translocatable tetracycline-resistance element Tn10. DNA/DNA heteroduplex and restriction enzyme analyses of alterations in the genome of bacteriophage lambda suggest that both types of DNA alterations almost always originate at the internal termini of the 1400 bp terminal inverted repetitions of Tn10. Tn10-promoted deletions remove a single contiguous DNA segment beginning at one such terminus; Tn10-promoted inversions are more complex, and involve both an inversion and a specific deletion of Tn10 DNA.


Nearly precise excision: a new type of DNA alteration associated with the translocatable element Tn10. We describe an unusual DNA alteration, "nearly precise excision," which has been identified among tetracycline-sensitive deletion derivatives of lambda phages carrying the translocatable tetracycline-resistance element Tn10. DNA sequence analysis of two such derivatives demonstrates that each retains exactly 50 bp of Tn10 material. The original junctions between lambda and Tn10 sequences remain intact; however, an internal deletion has occurred within Tn10 which eliminates all but the last few base pairs at each end of the element. This deletion occurs within a short A + T-rich inverted repeat which is present near each end of Tn10. Nearly precise excisions occur at frequencies comparable to Tn10-promoted deletions, inversions and translocations, and, like these other events, are independent of phage and bacterial functions for homologous recombination (recA, recB, red). It is not yet clear, however, whether nearly precise excisions are specifically promoted by Tn10 or whether they arise during the course of normal DNA replication processes as a consequence of unusual symmetries present in the DNA sequence at the ends of Tn10.


The chromatin structure of specific genes: II. Disruption of chromatin structure during gene activity. We have compared the chromatin structure in the active and inactive states at loci encoding the major heat shock protein in Drosophila. DNAase I and micrococcal nuclease were used as probes of higher order organization and nucleosomal integrity. Such integrity is gauged here by the characteristic pattern of discrete DNA fragments produced at specific chromosomal loci by nucleolytic cleavage. The specific fragment patterns are visualized by gel electrophoresis, Southern blotting onto nitrocellulose sheets, hybridization with 32P-labeled cloned DNA containing the heat shock genes and autoradiography. Using this criterion, a disruption in nucleosomal and possibly in higher order organization are observed as indicated by a relative loss or smearing of the characteristic discrete DNA fragment patterns from the heat shock loci in the active state. The fragment patterns are restored when cells are allowed to recover from heat shock and these loci return to the inactive state.


Four T7 RNA polymerase promoters contain an identical 23 bp sequence. The DNA sequences of four promoters for the bacteriophage-specified T7 RNA polymerase have been determined. All four regions contain an identical 23 bp sequence 5' pTAATACGACTCACTATAGGGAG-A(oh) which includes the 17 bp prior to and the first 6 bp of the transcribed region. They are located at 46.5, 55, 57 and 87% on the standard T7 map, thereby corresponding to the in vitro T7 class III RNA species IIIa, IV, V and IIIb, respectively. The precise RNA initiation sites were determined by 5' terminal RNA sequence analysis of the transcripts synthesized from promoter-containing DNA fragments. The comparative properties of these promoters for the monomeric T7 RNA polymerase and those for the multimeric E. coli RNA polymerase are discussed.


Sequence analysis of adenovirus DNA: complete nucleotide sequence of the spliced 5' noncoding region of adenovirus 2 hexon messenger RNA. The complete nucleotide sequence of the 5' noncoding region of the adenovirus 2 hexon messenger RNA has been established by sequence analysis of reverse transcripts. Such transcripts were generated by extension of specific single-stranded DNA primers with reverse transcriptase after hybridization to purified hexon mRNA. The total length of the 5' noncoding region was determined to be 240 nucleotides, of which the spliced tripartite leader sequence contributes 202 nucleotides including the terminal m7G. The sizes of the different segments of the tripartite leader were estimated by comparing the established mRNA sequence with the genomic sequences for the first and third leader segments, and were found to be 42 nucleotides for the first segment, 71 nucleotides for the second and 89 nucleotides for the third. The estimates are ambiguous, however, due to the presence of tandemly repeated sequences at both ends of the intervening sequence between the third leader segment and the body of the hexon mRNA. The sequence of the leader allows the formation of hydrogen-bonded interactions with the 3' end of 18S ribosomal RNA near the capped 5' end and also close to the initiator AUG.


Nucleotide sequence analysis of the leader segments in a cloned copy of adenovirus 2 fiber mRNA. Fiber mRNA of adenovirus 2 has been used as a template for RNA-dependent DNA polymerase. The resulting cDNA/RNA hybrids have been inserted at the Pst I site of the plasmid vector pBR322 after A:T tailing. One recombinant plasmid, pJAW 43, has been characterized in detail and shown to contain sequences from the main body of fiber mRNA, the three leaders common to most late adenoviral mRNAs and a fourth leader found in some species of fiber mRNA. The complete DNA sequence of the leader region has been determined and does not contain the initiation codon AUG, although this codon does occur immediately downstream from the junction between the fourth leader and the main body of the fiber mRNA. The first leader (map coordinate 16.6) is 41 nucleotides long, the second (from 19.6) is 71 nucleotides, the third (from 26.6) is 88 nucleotides and the fourth (from 78.5) is 181 nucleotides. The location of junctions between viral leaders and intervening sequences has been determined by reference, where possible, to sequences of the adenovirus 2 genome. Although the presence of short repeated sequences at the boundaries of intervening sequences and leaders makes it impossible to locate the splice point unambiguously, all of the leader-intervening sequence junctions can be arranged to stress a common feature--the presence of the dinucleotides GT and AG at the 5' and 3' ends, respectively, of the intervening sequences. This prototype sequence, which has also been recognized at or near the splice points in other eucaryotic systems, is possibly part of a larger unit which serves as a recognition site for specific excision-ligation events that ultimately lead to the production of mature mRNAs.


Microinjection of the nonhistone chromosomal protein HMG1 into bovine fibroblasts and HeLa cells. The nonhistone chromosomal protein HMG1 associated rapidly with the nuclei of HeLa cells and bovine fibroblasts following its introduction into the cytoplasm by red cell-mediated microinjection. A number of non-nuclear proteins, on the other hand, failed to concentrate in HeLa or bovine fibroblast nuclei. Autoradiography of thin sections showed that 125I-labeled HMG1 localized within nuclei, and further established that it remained associated with metaphase chromosomes at mitosis. When uninjected HeLa cells were fused with 125I-HMG1-injected HeLa cells, the labeled molecules equilibrated between nuclei within 12 hr. Similar results were obtained with bovine fibroblasts, indicating that a dynamic equilibrium exists between HMG1 and chromatin within living cells. Electrophoresis of 125I-HMG1 retrieved from HeLa cells or bovine fibroblasts up to 48 hr after injection showed that more than 80% of the molecules were intact. Autoradiographic analysis of cells fixed over a period of several days after injection produced apparent half-lives for 125I-HMG1 of 80 hr in HeLa cells and 100 hr in bovine fibroblasts.


Mammary plasminogen activator: correlation with involution, hormonal modulation and comparison between normal and neoplastic tissue. We have analyzed the plasminogen activator (PA) content of normal rodent mammary glands at different stages of the mammary life cycle and after exposing the animals to various hormones; we have also assessed the PA response of mammary explants to a variety of hormonal environments. Similar studies were performed on a limited number of primary mammary tumors. Plasminogen activator production was clearly correlated with mammary involution. A large but transient increase in enzyme content followed the initiation of involution in all glands, and the enzyme was produced by mammary cells, not by macrophages or granulocytes. Oxytocin, prolactin and hydrocortisone, which slowed or blocked involution, produced parallel effects on gland regression and PA synthesis. PA synthesis by explants in organ culture was induced by hormonal environments that fostered involution and repressed by those that promoted lactation. Mammary tumors produced much more PA than normal tissue both in vivo and in vitro, and distinct differences were found in the response of enzyme synthesis to hormones. The results reinforce the association of PA with tissue remodeling; show that the enzyme can be used as an indicator of cellular response to a wide range of hormones in both normal and malignant tissue; and suggest that observations of this type in organ culture may be of some value in predicting physiological responses in vivo.


The localization and synthesis of some collagen types in developing mouse embryos. The location of type IV (basement membrane)collagen in early post-implantation mouse embryos was examined by immunoperoxidase reactions using a specific immunoglobulin raised against mouse lens capsule collagen. Reaction was positive in the earliest embryos studied--on the fifth day of gestation (the day of detection of the copulation plug is the first day). It was found only in the primitive endoderm adjacent to the blastocoelic cavity. Subsequently in development, strong staining reactions were found in the parietal endoderm, Reichert's membrane and an acellular layer which separates the visceral endoderm of the egg cylinder from the ectoderm. In tenth to eighteenth day visceral yolk sacs, the mesodermal portion was stained, which is consistent with the presence of basement membranes around blood vessels. The endodermal portion of the visceral yolk sac did not react, while small amounts were found in the amnion. By incubation of various embryonic tissues with tritiated amino acids, purification of the biosynthesized secreted collagens and their partial characterization, the differential expression of several collagen genes was detected. Identification of collagen types was made by: reaction with specific antibodies to type I and IV collagens; electrophoretic mobility; sensitivity to reduction and to collagenase; analysis of the proportions of 3-hydroxyproline, 4-hydroxyproline and hydroxylysine; and CNBr peptides. In agreement with the data of Minor et al. (1976a) for the rat, mouse parietal endoderm synthesizes large amounts of type IV collagen. In contrast to their findings, however, the 165,000 molecular weight polypeptide is not converted to one of 100,000 after reduction, alkylation and repepsinization (Dehm and Kefalides, 1978). The endoderm of the visceral yolk sac was shown to be synthesizing primarily type I collagen, while the mesoderm layer of this membrane synthesized both type I and IV collagens. Little or no type IV collagen synthesis was detected in the endoderm of the visceral yolk sac. If it is correct that the visceral endoderm of the early embryo makes a major contribution to the formation of the endoderm portion of the visceral yolk sac, then it is clear that a switch in collagen gene expression must occur as it does so.


Two species of lysosomal organelles in cultured human fibroblasts. Cultured diploid human skin fibroblasts were fractionated by a procedure that maximizes recovery of particles containing acid hydrolases. The cells were detached by controlled trypsinization, disrupted by N2 cavitation at low pressure and fractionated at 18,000 x g on a self-generating gradient of colloidal silica. This procedure separated two species of particles that could be consisered lysosomal. The denser one (peak density 1.11) was apparently free of other contaminants, but the more buoyant one (peak density 1.085) sedimented with or close to the peaks of other organelles, including mitochondria, Golgi, endoplasmic reticulum and plasma membranes. The two populations of particles contained acid hydrolases (phosphatase, six glycosidases and four cathepsins) in roughly equal proportions, displayed latency, had similar turnover of 35S-mucopolysaccharide in normal as well as in iduronidase-deficient cells, and were recipients of alpha-L-iduronidase, previously shown to be acquired by receptor-mediated endocytosis. Acid phosphatase staining of the intact fibroblasts showed residual bodies scattered throughout the cytoplasm and, near the nucleus, a prominent network of tubules and associated dilatations and knob-like enlargements. In both thin and thick sections, these appeared continuous, as if forming a three-dimensional network similar to the network described by Novikoff (1976) as GERL. Ultrastructural studies of the isolated fractions showed the denser lysosomal peak to be composed of small round or oblong acid phosphatase-positive bodies. The more buoyant peak contained the nonlysosomal organelles predicted from the biochemical markers, small acid phosphatase-positive bodies and large multivesiculated structures in which acid phosphatase was localized in a matrix surrounding apparently empty vesicles. These large structures may represent fragments of GERL. We suggest that the dense and buoyant lysosomal organelles originate primarily from residual bodies and the GERL network, respectively.


Sequence organization of the spacer DNA in a ribosomal gene unit of Xenopus laevis. A detailed restriction map was constructed for a cloned Xenopus laevis rDNA fragment containing the nontranscribed spacer (NTS) and external transcribed spacer (ETS) together with a portion of both the 18S and 28S rRNA genes. The NTS was found to contain at least three distinct repetitious areas. Region 1 has a repeating unit of approximately 100 bp. The primary structure of this unit has been determined by DNA sequencing. Region 2 is very similar in organization to region 3, and both have an alternating 81/60 bp arrangement as revealed by restriction with Alu I and DNA sequencing. It can be shown that the 81 and 60 bp canons are virtually identical to one another excepting a deletion/insertion of a 21 bp segment. Region 3 differs from region 2 in having sites for Sma I with its 81 bp units. Between these repeated DNA sequences there are two identical, nonrepetitive DNA sequences, each of which is centered around a Bam Hl site. Most of the ETS has been sequenced. It was found to be nonrepetitive and extremely rich in Cs. Close to the 5' end of the 18S coding sequence there is a DNA stretch very rich in purines. About 2.25 kb upstream from the Eco Rl restriction site bisecting the 18S structural gene there is a unique sequence which may be homologous to the 5' end of the 40S precursor RNA. Present evidence suggests that the boundaries between NTS and ETS occur farther downstream than was suggested by electron microscopic data. Sequencing has revealed that the spacer DNA of X. laevis contains different kinds of simple DNA sequences, but no evidence has been found that spacer DNA once arose by saltation of a 15 bp segment. The most surprising finding was that the spacer sequences around the Bam restriction sites (the Bam islands) show high homology with a sequence near the NTS/ETS interface. From the restriction and sequencing analyses it can be deduced that in recent evolutionary times the DNA sequences near the 5' end of the ribosomal transcription unit were reduplicated twice and displaced into spacer by saltation of an intervening short DNA sequence (the 60/81 bp canons). Possible implications of these evolutionary events for spacer functions are consisdered. The sequencing has also provided a molecular basis for a whole range of conclusions arrived at previously by indirect approaches, and these are discussed.


The molecular basis of alpha-thalassemias: frequent occurrence of dysfunctional alpha loci among non-Asians with Hb H disease. Study of Asians has previously indicated that deletion of alpha-globin structural genes is the predominant lesion in alpha-thalassemias and that Hb H disease occurs when three of four normal alpha loci per cell are deleted. To test the generality of this model, Hb H disease DNAs of both Asian and non-Asian origin were analyzed by restriction endonuclease mapping using the technique of Southern (1975). Whereas in normal DNA, alpha sequences are present in a single Eco Rl fragment of cellular DNA approximately 22.5 kb long, fragments of 22.5, 20 and 2.6 kb were found in various Hb H disease DNAs. The 20 kb Eco Rl fragment alone, in which a single alpha-globin structural locus resides, was found in Asian Hb H disease DNA. This finding is consistent with the deletion model of alpha-thalassemia. In contrast, seven of eight non-Asian Hb H disease DNAs displayed a more complex molecular composition. The fragment patterns observed were 22.5 kb alone, 22.5 plus 2.6 kb, 20 plus 2.6 kb and 20 kb alone. Non-Asian Hb H disease DNAs contained one, two or three alpha loci per cell in contrast to the one locus predicted by the simple deletion model of alpha-thalassemia. The data are best explained by the existence of defective alpha loci in certain individuals with alpha-thalassemia, particularly outside the Asian population. Restriction mapping of the 20 kb Eco Rl fragment found in Asian and some non-Asian Hb H disease DNAs demonstrated a striking similarity in the placement of restriction sites about the single alpha gene compared with sites about the two genes in the 22.5 kb Eco Rl fragment seen in normal DNA. These data are consistent with origin of the 20 kb fragment from the 22.5 kb normal Eco Rl fragment by either unequal crossing-over or a deletion event. The molecular heterogeneity and frequent occurrence of defective alpha loci in non-Asian Hb H disease DNAs described here may explain, in part, the clinical heterogeneity of alpha-thalassemias and the absence of the homozygous deletion state (hydrops fetalis) in non-Asians. Further study of cellular DNA fragments containing the defective alpha loci identified in this work may indicate the types of specific mutations responsible for abnormal globin gene expression and complement similar studies on abnormal beta genes in beta-thalassemias.


Manipulation of myogenesis in vitro: reversible inhibition by DMSO. A system has been developed for the detailed analysis of the transition from proliferative myoblast to differentiated muscle cell. Dimethylsulfoxide (DMSO) prevents the terminal differentiation of L8 myoblasts in vitro, and its effect is reversible. DMSO (2%) inhibits the fusion of myoblasts to form multinucleate myotubes, the normal increases in activity of creatine phosphokinase (CPK) and acetylcholinesterase, and the synthesis of alpha-actin and acetylcholine receptor protein. Upon removal of DMSO from the medium, a lag precedes the onset of differentiation. The potential to inhibit muscle differentiation reversibly is not specific to DMSO, but is shared by a number of compounds, including dimethylformamide, hexamethylbisacetamide and butyric acid, all potent inducers of gene expression in Friend erythroleukemia cells. L8 cells routinely cease DNA synthesis and initiate fusion and muscle protein synthesis once they are confluent. In the presence of DMSO, however, nearly all cells continue DNA synthesis, even several days after reaching confluence. Protein synthetic patterns of DMSO-inhibited cells are almost indistinguishable from those of untreated myoblasts and distinct from differentiated myotubes. It appears that cells exposed to DMSO are locked indefinitely in a proliferative myoblast stage of development and are unable to enter the Go phase of the cell cycle necessary for initiation of differentiation. DMSO coordinately inhibits all the differentiative parameters measured. In contrast, cytochalasin B uncouples normally linked differentiative events so that fusion is inhibited while muscle-specific protein synthesis proceeds. DMSO has similar effects on both cytochalasin B-treated and fusing control cultures, suggesting that its primary effect is exerted not at the level of fusion but earlier in the differentiative time-table. Once fusion and the synthesis of muscle-specific proteins are well under way, the addition of DMSO is ineffective and differentiation continues in its presence. The potential to manipulate muscle gene expression in vitro makes this system particularly useful for the detailed analysis of the processes involved in the transition to the differentiated state and for determining the linkage of developmental events.


Fibronectin mediates cytokinesis and growth of rat follicular cells in serum-free medium. In this study, the growth of rat follicular (RF-1) cells was severely depressed when the cells were subcultured by trypsinization directly into serum-free medium supplemented with insulin, transferrin and hydrocortisone, which are required for growth of these cells in vitro. Within 24 hr after plating, 50-65% of the cells became binucleated, indicating lack of cytokinesis. However, the addition of human plasma fibronectin (8 microgram/ml) to the serum-free medium eliminated cell binucleation and enhanced cell growth considerably. Fibronectin had the same effect when RF-1 cells were plated into tissue culture dishes on which fibronectin had been absorbed, and cells were inoculated into fibronectin-free medium. Cell binucleation and poor growth in serum-free medium occurred when the cells were subcultured by trypsinization, EGTA treatment or detachment of mitotic cells. Under some conditions, cells could be "rescued" if fibronectin was added soon after inoculation, indicating that fibronectin was needed mainly during a limited time when the subcultured cells were attaching to the tissue culture substratum. Our findings describe an adhesive activity for fibronectin which circumvents the serum preincubation usually needed after RF-1 cells are subcultured for growth in serum-free medium. They also indicate the importance of fibronectin-mediated adhesion for cytokinesis processes of these cells.


Cilia regeneration in starved tetrahymena: an inducible system for studying gene expression and organelle biogenesis. Deciliated starved Tetrahymena recover motility with kinetics similar to those of growing cells and, like growing cells, require RNA and protein synthesis for regeneration. Comparisons of polysome profiles and electrophoretic analyses of newly synthesized proteins indicate, however, that the basal level of protein synthesis in starved cells is markedly lower than that in growing cells. This difference allows demonstration of changes in protein synthesis following deciliation of starved cells which cannot be detected (if they occur at all) in growing cells. Deciliation of starved cells induces a specific and orderly program of protein synthesis. The synthesis of an 80,000 dalton protein (deciliation-induced protein, DIP) begins shortly after deciliation, comprises 15% of the protein synthesized from 20-60 min, and declines around 60 min after deciliation, shortly after most cells have begun to regenerate cilia. The synthesis of a 55,000 dalton protein is also induced during regeneration and has been identified as tubulin using a well characterized antibody made to ciliary tubulin. Tubulin synthesis is undetectable during the first hour after deciliation even though 60-80% of the cells regain mobility and regenerate short but clearly visible cilia. Tubulin synthesis begins 60 min after deciliation and continues for 2 hr. At its peak, tubulin comprises 7-8% of the protein synthesized. The results of actinomycin D addition at different times after deciliation suggest that RNA required for DIP synthesis is synthesized early (0-30 min), while RNA required for tubulin is synthesized later and over a longer period (30-90 min). Thus deciliation of starved cells, an event occurring at the cell periphery, initiates a well defined and reproducible series of events culminating in cilia formation. This system should be useful in elucidating the molecular mechanisms regulating gene expression and organelle biogenesis in Tetrahymena.


Mechanisms of regulating tubulin synthesis in cultured mammalian cells. Colchicine and nocadazole both depolymerize microtubules in cultured fibroblasts and lead to a rapid inhibition of tubulin synthesis. The level of translatable tubulin mRNA is greatly reduced in drug-treated cells as demonstrated by translation in a reticulocyte-derived in vitro protein synthesizing system. A model of tubulin synthesis regulation is proposed in which the elevated level of unpolymerized tubulin in drug-treated cells inhibits the formation of new tubulin mRNA and the preexisting message decays rapidly. In agreement with this model, tubulin message is found to be short-lived and has an approximately 2 hr half-life in cells treated with actinomycin D. Another prediction of the proposed model is that destabilization of microtubules without a concomitant increase in free tubulin will not inhibit tubulin synthesis. Vinblastine also disrupts microtubules but leads to the aggregation of tubulin into large paracrystals with an apparent decrease in the concentration of free tubulin. This drug does not inhibit tubulin production but rather leads to a measurable enhancement of tubulin synthesis.


H-Y binding in the gonad: inhibition by a supernatant of the fetal ovary. H-Y antigen, the product of mammalian testis-determining genes, is released in the free state by testicular cells. Molecules of free H-Y antigen are bound in vitro by dispersed cells of the adult ovary. The binding reaction is inhibited by specific H-Y antibody. It is also inhibited by a diffusible factor of the newly differentiated fetal ovary. These observations favor the view that testicular organogenesis depend upon dissemination and binding of H-Y molecules by cells of the undifferentiated gonad (XY or XX, both having H-Y receptors), and raise the question of whether ovarian organogenesis may be promoted by a "female" molecule corresponding to H-Y of the male.


Reiteration frequency of the gene coding for the small subunit of ribulose--1,5--bisphosphate carboxylase. The messenger RNA coding for the precursor of the small subunit of ribulose--1,5--bisphosphate (Ru-P2) carboxylase has been partially purified from pea leaves. The mRNA has a size of 11.5S, is approximately 800 nucleotides long and, on cell-free translation, directs the synthesis of a single major polypeptide of 20,000 daltons. Antiserum for the small subunit of Ru-P2 carboxylase immunoprecipitated 52% of the 35S--methionine-labeled cell-free translation products. The RNA hybridizes with pea DNA with monophasic kinetics corresponding to the presence of one, or very few, gene copies per haploid genome.


Alleles of the fibroin gene coding for proteins of different lengths. Bombyx mori silkworms producing fibroin proteins of different lengths have been analyzed genetically and shown to possess variant alleles of a single fibroin gene. The structures of two alleles have been compared by using restriction endonuclease sites inside and outsite the fibroin gene as physical markers. We find that fibroins distinguishable on the basis of length are encoded by genes with different internal structures and overall lengths. Our results strongly support the idea that rearrangements within the highly repetitive sequences of the fibroin gene are the result of unequal recombination, and can give rise to variant fibroin genes with altered coding lengths.


An abundant androgen-regulated mRNA in the mouse kidney. We have identified an abundant 20,000 dalton protein (KAP) by in vitro translation of male mouse kidney mRNA. This protein is synthesized in reduced amounts from female kidney mRNA. A KAP cDNA fragment was purified and used for nucleic acid hybridization studies. Females and castrated males have 10 and 200 fold lower levels, respectively, of KAP mRNA relative to males. The administration of testosterone to females or castrated males results in the induction of KAP mRNA to normal male levels. Testicular feminized (Tfm) mice have 3 fold lower levels of KAP mRNA relative to normal males and are not induced by testosterone. KAP mRNA is not found in significant amounts in tissues other than the kidney, and the KAP gene renatures with kinetics similar to single-copy DNA. With the rapidly expanding knowledge of mouse genetics, KAP should prove useful in determining genetic factors which regulate the inducibility and tissue specificity of a hormonally regulated gene.


Role of prolactin the the expression of casein genes in the virgin rabbit. Prolactin was injected into mature virgin rabbits. Casein mRNA, already present prior to the treatment was enhanced by prolactin. This accumulation of casein mRNA was not accompanied by a parallel significant synthesis of casein. Th effect of prolactin took place without a previous cellular multiplication. The mammary cell is therefore sensitive to prolactin before the first pregnancy but prolactin then displays only an abortive effect on the expression of casein genes.


Protein synthesis and differentiation in a clonal line of teratocarcinoma and in preimplantation mouse embryo. Undifferentiated cells of a clonal line of teratocarcinoma can differentiate in vitro into embryoid bodies with morphological and biochemical features of early mouse embryo. During the first step of differentiation protein synthesis has been analysed by 2 dimensional gel electrophoresis. While new proteins are synthesized, the synthesis of others turned off with the appearance of endodermal cells in embryoid bodies. We have compared protein synthesis during teratocarcinoma differentiation and during early mouse embryogenesis at three stages of mouse preimplantation embryo. The results demonstrate that only the late blastocyst protein synthesis pattern shows most of the polypeptides identified in the differentiated protein synthesis pattern of teratocarcinoma. In contrast, protein synthesis during the early stages of mouse embryonic development is very different from protein synthesis in undifferentiated teratocarcinoma.


Cell proliferation in the mouse parotid gland after unilateral parotidectomy. Unilateral parotidectomy with or without total submandibulectomy has been used to induce cell proliferation in mouse parotid gland. Maximum DNA synthesis and mitosis were recorded four and five days after the operation. The double operation increased the proliferative response. Such proliferative stimulii was not accompanied by secretion and was sex independent. On the other hand, the response decreased with age. RNA and protein synthesis inhibitors showed that the stimulation of DNA synthesis depends on early protein synthesis, which seems to be synthesized on a preexisting template.


Activation of lymphocytes by concanavalin A requires calcium ions. Mouse spleen cells were exposed to a short pulse of the mitogenic lectin concanavalin A (con A). After removal of con A mitogenesis was measured by the incorporation of tritiated thymidine into DNA. It was found: (a) the number of cells responding to con A was proportional to the time of exposure to con A; (b) exposure of cells to con A in the absence of extracellular calcium failed to initiate mitogenesis; (c) for a mitogenic effect an extracellular calcium concentration greater than 10(-5)M was required during the time that the cells were exposed to con A.


Diminution of L cell microvilli following exposure to 25-hydroxycholesterol. The effect of sterol depletion on the topology of mouse L cells was studied by scanning electron microscopy. Treatment of cells with 25-hydroxycholesterol inhibited the synthesis of cellular sterol and diminished the number of microvilli on the cell surface. Simultaneous addition of mevalonic acid or cholesterol to cells during treatment with the inhibitor prevented the loss of microvilli. These results demonstrate that cholesterol is important in maintaining the ultrastructure of the surface membrane of nucleated mammalian cells.


Tetrahymena cells secrete a low-molecular weight factor which inhibits RNA synthesis in vivo and in vitro. Medium which has been dialyzed against a dense population of Tetrahymena cells contains a factor which reduces the rate of RNA synthesis in other Tetrahymena cells inoculated on that medium. Furthermore, this extracellular factor inhibits the ribosomal RNA synthesis in vitro when added to an RNA synthesizing r-chromatin preparation.


On the occurrence and significance of annulate lamellae in gastrodermal cells of regenerating planarians. Cytoplasmic annulate lamellae have been observed to occur only in a subset of the gastrodermal cell population of regenerating planarians. They have not been found in the gastrodermal cells of intact, non-injured worms, nor in any other somatic cell type. These observations plus the presence of numerous chromatoid bodies in the same cells are consistent with the hypothesis that these cells are altering their state of differentiation and are preparing for division. It is further suggested that these cells are the precursors to the definitive somatic stem cells, the beta cells.


Chloride permeability of sea urchin eggs. We have examined the content and permeability of chloride in sea urchin eggs. After fertilization there is a large increase in the permeability to chloride. We discuss the mechanism underlying this permeability change and the generalized increase in ion permeability observed after fertilization.


Development of the sarcotubular system in fusion-arrested myoblasts. We have compared the effect of two different procedures, equally effective in preventing muscle cell fusion in culture, on the development of the sarcotubular system in rat muscle cells. Whereas in myoblasts grown in low Ca++ medium the T system was poorly developed and diadic or triadic couplings between T tubules and sarcoplasmic reticulum were rare, in cytochalasin B-treated myoblasts the development of the sarcotubular system was comparable to that seen in myotubes of the same age. We conclude that (a) muscle cell fusion is not essential for the development of the sarcotubular system, and (b) procedures used to prevent cell fusion in vitro may affect directly muscle cell differentiation by a process independent of the fusion block.


Association of human chromosome 14 with a ts defect in G1 of Chinese hamster K12 cells. Somatic cell hybrids between human lymphoblastoid cells (Raji) and temperature-sensitive Chinese hamster cells (K12) were selected from monolayer cultures in MEM at 40 degrees C. A total of 21 hybrid clones were isolated and karyotyped. All clones contained a near complete set of Chinese hamster chromosomes and 1 to 5 human chromosomes. Human chromosome 14 present in the hybrid cells of all clones; and was the only human chromosome retained in 10 clones. The presence of human chromosome 14 in hybrids was further confirmed by the demonstration of human nucleoside phosphorylase activity in the hybrid cells. Only one hybrid clone was positive for EBNA, the Epstein-Barr virus antigen present in Raji cells. These findings indicate that human chromosome 14 contains the necessary information for the K12 cells to overcome their G1 defect in the cell cycle and grow at non-permissive temperature. The present study lends strong support to the possibility that different steps in the G1 phase of the cell cycle are controlled by genes located on different chromosomes.


[Edwards' syndrome].

[Edwards' syndrome]. Two cases of Edwards' syndrome were observed. One occurred in a girl aged 6 months, the other in a male neonate. The two children showed a highly characteristic appearance of both the face and the extremities, so that it arouse suspicion of 18-chromosomy. The latter was confirmed by a postmortem cytogenetic examination. The syndrome also includes malformations of inner organs and microscopical architectural changes of organs, which occurred in both the cases described, but different in type.


[Cystic degeneration of adventitia].

[Cystic degeneration of adventitia]. A description is presented of the cystic degeneration of the adventitia of the external iliac and femoral arteries. Numerous cysts in the adventitia compressed the arterial lumen and resulted in thrombotic occlusion. The histologic picture was similar to that of tendinous sheath ganglion. A list is given of cases already described with a discussion of the views concerning its aetiology and pathogenesis.


[Solid dendritic angiosarcomas (of the hemangioendothelioma and hemangiopericytoma types in the differential diagnosis of malignant lymphomas)]. A group of 11 cases was used for the closer follow-up of an extraskeletal tumour lik Ewing's sarcoma (9). While mostly soft tissues were affected, its localization was variegated. Throughout the long-term follow-up period there was only one case of non-relapse after extirpation. Most of the tumours became generalized within three years. One exceptional case involved generalization after 13 years. The tumour reveals a mostly solid, seemingly organoid pattern, always, however, with reticular foci or sinusoid in nature. The cells, round or dendritic in appearance, have numerous intercellular connections with Weibel Palade granules occasionally demonstrable in their cytoplasm. In foci of peritheliomatous appearance, such as may exceptionally predominate in the tumour, the tumour cells become elongated, contain an abundance of endoplasmic reticulum, and make up a system of integrated basal membranes. According to its structural and cytological properties, the tumour has the nature of angiosarcoma, mostly of the endothelial type, frequently assuming the nature of the pericytic variant. These transitional structural properties just as the tumour' biological behaviour are unlike Ewing's sarcoma and, for that reason, a change of the name is proposed.


[Clear-cell carcinoma of the thyroid gland]. The case of a 66-year old woman is presented involving the partial transformation of follicular carcinoma with a solid somponent into clear-cell carcinoma of the thyroid gland. The carcinoma in its follicular component was seen invading the capsule, striated muscle, and vessels, and metastasizing in the form of a solitary metastasis into the ilium. Postmortem examination ruled out any other aetiology of the clear-cell carcinoma.


[Immune complexes in sudden and violent deaths]. Immunocomplexes in the blood serum of 115 autopsied subjects chosen at random were examined by means of the Pegikem test. Average values of 34.9 +/- 44.6 were found in a group of 57 suddenly died subjects while those of 51.3 +/- 76.2 were seen in a group of 58 subjects dead due to injury. The possibility of the determination of immunocomplexes for postmortem diagnosis of immune conditions are discussed.


[Indroduction of minicomputers into instrumental chromatographic technics]. Research in and development of computer techniques and large-scale integration technology have made considerable progress over the past thirty years. Examples of the development of gas chromatography, highly effective liquid chromatography and mass spectrometry apparatures are given to show the most up-to-date equipment enabling, in combination with computer tecniques, modernization, automation and economical retrieval and processing of the results of investigations. The authors discuss the uses of microcomputers, in particular the so called scientific programming pocket calculator and their anticipated contribution. There are also examples of automatic apparatus application, particularly those for therpay monitoring, i.e. primarily the estimation of the levels of certain highly effective drugs, or drugs with undesirable side-effects (e.g. diphenylhydantoin drugs, barbiturates, aminoplvyllin, etc.).


Binding of beryllium to nuclear acidic proteins. In vitro beryllium (Be) binding to rat liver nuclei has been reassessed (KAss = 2.0 X 10(6) M: n = 17 nmol Be/mg protein). Be also binds to rat liver nucleoli (KAss approx. 4 X 10(6) M: n = 10 nmol Be/mg protein). Examination of rat liver chromatin fractionated on a hydroxyapatite column shows that Be does not bind to histone or to the non-histone protein eluted by 0.05 M sodium phosphate. Be is strongly bound to the non-histone proteins eluted by 0.2 M sodium phosphate (KAss = 1.1 X 10(6) M: n = 55 nmol Be/mg protein) and also to the same extent to the fraction containing DNA which is subsequently eluted from the column. Evidence is provided that the latter binding is not due to DNA. The fractions containing the Be-binding proteins also contain the proteins which are phosphorylated to the greater extent.


The interaction of aquated cis-[(NH3)2Pt(II)]with nucleic acid constituents. II. Modified nucleosides. The binding interaction of aquated cis-(NH3)2Pt(II) with 1-methylguanosine (1-MeGuo), 7-methylguanosine (7-MeGuo), 1-methyladenosine (1-MeAdo+) and protonated adenosine has been studied using UV difference spectroscopy. The magnitude of the binding constants for the 1 : 1 interaction of cis-(NH3)2Pt(II) with 1-MeDuo and 1-MeAdo are log K = 3.5 and 3.7, respectively. These data are presented and compared to other cis-(NH3)2Pt(II)-nucleoside interactions.