ALBERT

Notes: Early in conjugation, both in micronucleate and amicronucleate ciliates, the original oral structures degenerate and are quickly succeeded by new rudiments which may or may not attain functional competency. These changes were followed in matings of amicronucleate Oxytricha fallax and, in somewhat less detail, of amicronucleate Paramecium multimicronucleatum and P. trichium.In the ex-conjugants of Oxytricha the new peristomes did not reach complete structural development: they bore a deficient number of adoral membranelles and lacked an undulating membrane along the right border. Numerous isolated pairs gave rise to ex-conjugants, all of which died within a few days. Macronuclear changes in conjugation seemed normal.In P. multimicronucleatum most of the ex-conjugants lacked a buccal cavity. Either the second generation oral apparatus had failed to develop or had disappeared quickly after it did arise. An occasional ex-conjugant possessed an oral opening which could have been the persisting original buccal cavity, or a possible new one. Isolated pairs yielded 80-100% lethality. The discrepancy from 100% is explainable on the basis of precocious separation of a small number of pairs before their macronuclei had reached irreversible skein formation, as judged by stained specimens. The macronuclear changes were somewhat different from those of micronucleate conjugation.P. trichium amicronucleate conjugations were studied only from cases in mass cultures, all of which died out. Although most of the ex-conjugants lacked buccal cavities, there was a higher percentage of individuals with mouths than was true for P. multimicronucleatum.

Notes: RNA synthesis in isolated Drosophila salivary glands was studied by autoradiography. The dry mass of cellular structures was determined by interference microscopy; a correction which compensates for self-absorption of β-particles, and consequent lowered grain counts, is discussed. The correction factors are 12.0 for nucleolus, 5.0 for nucleoplasm and 8.8 for cytoplasm.After 15 seconds in uridine-H3, label was localized over the nucleolus; after one minute, nucleoplasm RNA became labeled also. In situ synthesis of some nucleolar RNA is indicated.Comparisons of uridine-H3 and cytidine-H3 incorporation were made in glands exposed briefly to isotope, followed by transfer to unlabeled nucleoside solutions. The data suggest two RNA fractions in nucleoli. Rapid turnover and higher uridine incorporation characterized one fraction, which resembled RNA of nucleoplasm metabolically. The second nucleolar RNA incorporated less uridine, remained longer in the nucleolus, and resembled cytoplasmic RNA in precursor incorporation pattern.Preincubation in Actinomycin D before uridine-H3 labeling resulted in 80% inhibition of RNA synthesis in both nucleolus and nucleoplasm. Longer isotope exposures produced some increase in labeling. Actinomycin treatment delayed appearance of label in cytoplasmic RNA.After HCl extraction of uridine-H3-labeled RNA, some activity, presumed to be incorporated in DNA, remained. This non-extractable label appeared earliest over nucleoli, and subsequently over chromatin. Actinomycin treatment abolished incorporation of non-extractable label into nucleoli.

Notes: At or shortly prior to the separation of the cuticle and the epidermis a cuticular layer appears to be added to the inner surface of the old cuticle. The term interzone cuticle is applied to this layer. The interzone cuticle appears to give rise to the ecdysial membrane.Ecdysial membranes are characterized by the fact that they (1) vary in position with respect to the moulting fluid and old cuticle, (2) are generally quite thin, transparent, delicate membranes, (3) contain chitin, (4) vary in their resistance to the action of the moulting fluid, and (5) generally have the same stainng properties as the subcuticle, due primarily to the presence of a mucous material present in the subcuticle which becomes attached to the inner surface of the ecdysial membrane and which is believed to function as a lubricant at ecdysis.The ecdysial membrane and subcuticle are structurally quite different when examined under the electron microscope.No particular evolutionary significance can be ascribed to the existence of an ecdysial membrane  -  such membranes being found all the way from the primitive Collembola to the advanced Hymenoptera. Any possible function(s) of the ecdysial membrane has yet to be discovered. Various functions are suggested for the subcuticle, all of which are consistent with its reactions to various histochemical reagents.Additional observations were made and discussed concerning (1) the reactions of various portions of the cuticle at various stages to the PAS test, (2) the staining reactions of cuticle undergoing enzymatic degradation by the moulting fluid, and (3) the distinct staining reactions of the intersegmental membranes and basement membranes.

Notes: The dog mandible has three joints  -  two temporomandibular and one symphyseal joint. In studies of jaws and their mechanics usually only the temporomandibular joints are considered. The symphysis is consistently neglected and because of this, remarkable features of the temporomandibular joints have never been noted.The symphysis has special connective tissue arrangements and its own vascular and nerve supply. Along the anterosuperior part of the symphysis, a strip of fibrocartilage, and running more or less parallel to it, strong cruciate ligaments bind the symphyseal plates together. Small arteries run into the symphyseal joint and it is drained by an anastomosing venous plexus. The posterior, ligamentous, portion is laced with a profuse network of nerves.The temporomandibular joint is unusual in that its joint disc is powerfully anchored to the skull medially and to the outer pole of the mandibular condyle laterally. The temporomandibular joint disc thus acts as a ligament that halts lateral movement of the condyle just as the carnassial teeth are aligned for cutting function. As the mandible moves laterally, the space between the posterior ends of the symphyseal plates widens: at the same time it narrows anteriorly. The cruciate ligaments control this movement and stabilize the symphyseal joint by preventing direct lateral displacement of the symphyseal plates. The fibrocartilage cushions this movement and also mediates rotational movements about the longitudinal axis of the mandible when crushing molar teeth come into function.

Notes: Interferometric measurements were made on sections of salivary glands mounted in oils of refractive indices 1.410, 1.460, and 1.500, estimating optical path differences as fringe displacement with a Leitz double-beam interferometer microscope. From these data the mean concentrations (in mg/cm2/micron) of 55 cells of three glands were computed as follows: karyoplasm, 0.027±0.0026; cytoplasm, 0.048 ± 0.0024; nucleolus, 0.066±0.0041. From these concentrations it was computed, according to Maurer and Primbsch (Exp. Cell Res., 33: 8-18) that the correction factors for four micron sections were: karyoplasm, 0.203; cytoplasm, 0.114; nucleolus, 0.083. The reciprocals of these fractions (5.0, 8.8, and 12.0) are the correction factors of Arnold (J. Morph., in press) by which measured silver grain densities on autoradiographs are multiplied to give corrected (i.e. potential) densities.

Notes: Glycogen, determined by Best's carmine and/or PAS Tripas staining, and alkaline phosphatase, determined by the Gomori technic, occur together throughout the epithelium of the genital tract. Sites of high alkaline-phosphatase activity are the acinar epithelium, nurse cells, and follicular cells of the ovotestis. Sperm heads are also strongly positive for the enzyme. The nidamental gland cells and the subepithelial mucous cells associated with the loop of the hermaphroditic duct and with the spermatic groove are thionine metachromatic, alcianophilic, PAS-positive and amylase-fast. The mucous cells of the genital atrium are PAS-positive and amylase-fast but lack both thionine metachromasia and affinity for Alcian blue.

Notes: The quinoidal secretion, secreted by holocrine action from most gland cells (no cytoplasmic inclusions), of Orthocricus arboreus may ooze out or be sprayed over 30 cm. A thick gland intima and basement membrane protects internal organs. The gland grades into a neck leading to an external orifice. An ejaculatory duct, its lumen closed by cuticular spring action, is formed by invagination of the neck. Part of the duct forms a tongue, which can independently unplug the orifice. Retractor muscles open the ejaculatory duct, secretion flows in from the gland, and the part of the duct nearest the gland closes. Sequential relaxation of the rest of the retractor muscles build up fluid pressure, which results in a spray when the orifice is unplugged. A ligament opposes the pull of the retractor muscles. Opening both duct and orifice results in secretion ooze. No intrinsic gland muscles were found. Intersegmental muscles may produce hemolymph fluid pressure on the gland, thereby contributing to secretion ooze. Illustrations are presented. A table comparing the two basic types of millipede repugnatorial glands (spirobolid and polydesmid), on the levels of structure, histology, secretion, chemistry and function is presented. The polydesmid type (2-chambered) probably evolved from the spirobolid type (1-chambered). The secretion of one type would not work in the other type of apparatus. A second table corelates the secretion and ejection mechanism of 23 species of millipeds with their phylogenetic position in the class Diplopoda.

Notes: Tissues of White Leghorn embryos of stages 17-45, and chicks of one day, two days, and three weeks of age were frozen or fixed in 85% ethanol. Alkaline phosphatase was identified using the azo-dye or Gomori techniques.Alkaline phosphatase activity is found in the ventral mesenchyme of the esophagus surrounding the epithelium to stage 34. At stage 38 activity appears in the basal layers of the epithelium and is subsequently lost in the mesenchymal tissues. This activity persists in the epithelium throughout development and following hatching. At 16 days the mucous glands arise as solid buds of epithelium projecting into the tunica propria. These buds are highly positive for alkaline phosphatase. As differentiation of the glands becomes complete they and their ducts lose their positive phosphatase reaction.On the fourth day of development the trachea is found lying ventral to the esophagus. Mesenchyme has condensed around the tracheal epithelium by the fifth day, at stage 37 (11 days) the first cartilaginous rings appear, and by stage 38 (12 days) all are completely formed. During the third week of development the trachea increases in size. Pseudo-stratified columnar ciliated epithelium begins to appear at 15 days. Alkaline phosphatase is highly reactive in the mesenchyme surrounding the epithelium prior to cartilage formation. After the cartilages have formed the reaction diminishes and in late stages of development phosphatase is found only in the epithelium.

Notes: An investigation of the innervation of the hand muscles of the opossum, Didelphis marsupialis virginiana, Kerr, was made to determine the pattern of hand muscle innervation in a relatively primitive quadruped mammal. Four pairs of preserved opossum hands were dissected, and the innervation of their muscles studied with a stereobinocular microscope. The distributions of the median and ulnar nerves to the hand muscles are outlined, and the details of distribution are illustrated by means of seven diagrams.Peculiar anastomoses were consistently found between the superficial interdigital rami of the median nerve and branches of the deep volar ramus of the ulnar nerve, and the short deep flexors of digit II consistently received all, or a part, of their innervation from superficial interdigital rami of the median nerve. Five features of these unusual nerve connections and distributions are summarized and discussed.

Notes: The primitive structure of the salamander otic capsule (exemplified by Hynobius) includes both operculum and columella in the fenestra vestibuli and connection of operculum with suprascapsular cartilage by an “opercularis” muscle formed by levator scapulae. Larvae of Hynobiidae, Ambystomatidae, and Salamandridae have early development of columella and stilus, which are used in sound transmission via the suspensorium until the development of the operculum and its muscular connection with the shoulder girdle at metamorphosis. Hynobiids and ambystomatids show parallel trends toward reduction or fusion of the operculum and widening of the attachment area of the levator scapulae on the suprascapular cartilage, and some members of both families have lost the operculum.The Salamandridae have lost columella and stilus as adult structures, but extend the attachment of levator scapulae ventrally by a division resulting in attachment to the scapulocoracoid, probably as an adaptation for better reception and transmission of sound vibrations in terrestrial environments.The Plethodontidae differ strikingly in otic structure from the other families in having an operculum present in the larval stage and fused to columella, and in utilization of part of the cucullaris major as an “opercularis” muscle. These changes are probably the result of changes in orientation of the fenestra vestibuli. In plethodontids with aquatic larval stages the columella is retained as a sound-transmitting element, but in many specialized forms it is reduced or absent.Close correlation of otic structures with ecology limits their use as evolutionary indicators in salamanders. However, the general pattern of otic structures in salamanders, and the magnitude of differences in this region seen in plethodontids, indicates that the latter are probably not derived from the salamandrids, but from a primitive salamander stock.

Notes: Observations on chelonian intracranial arteries are based primarily on the adult condition in Pseudemys scripta and on a series of Chrysemys marginata embryos. Those vessels which show major modifications are the anterior cerebral, posterior cerebral, cerebellar, and internal auditory arteries. The distal portion of the embryonic anterior cerebral is acquired by more medial vessels; from the proximal portion develops the middle cerebral which becomes the major source of supply for the lateral surface of the adult cerebral hemisphere. The posterior cerebral appears early in development and eventually supplies branches to the epiphysis, posterior portions of the hemisphere, olfactory regions, anterior face of the optic lobe, and choroid plexus of the third and lateral ventricles. The cerebellar and internal auditory arteries show similarities in development, each initially supplying the area immediately surrounding a nerve root and then acquiring larger areas of distribution by means of anastomoses with nearby medullary vessels.

Notes: The histochemistry of the “sexual segment” granules of the kidney of male diamondback rattlesnakes has been studied to define the nature of these andro-genically responsive granules. The kidneys were variously fixed and sections stained with a number of acid dyes, as well as by a variety of carbohydrate, lipid, and protein histochemical methods. The results indicate that “sexual segment” granules bind acid dyes strongly, contain some lipids and neutral glyco- or mucoproteins, much tyrosine and lysine, and some tryptophan and cysteine. A resemblance, concluded to be superficial, is noted between the histochemical properties of zymogen granules and the predominantly proteinaceous “sexual segment” granules.

Notes: The development of the chondrocranium of the Egyptian snake, Psammophis sibilans has been thoroughly studied in six developmental stages. The neurocranium and viscerocranium develop at the same time. In early stages the mesocephalic flexure is well represented, but later it is gradually reduced. The notochord is completely embedded inside the parachordal plate in the early and moderate stages, and the paired origin of the plate is not observed. From the very beginning the quadrate cartilage is in close contact with Meckel's cartilage. In early stages the two cartilages lie nearly at the same straight line, and throughout development the quadrate rotates freely until the angle between it and Meckel's cartilage becomes nearly a right one. It was impossible to find a stage at which the auditory capsules and their cochlear portions were separate from the parachordal plate. There is no interorbital septum and the platytrabic character of the chondrocranium is preserved. The basicranial fenestra, fenestrae X and the fenestra ovalis are formed by the resorption of pre-existing cartilage. The planum antorbitale has no separate center of chondrification. The rudiment of a simplified type of concha nasalis is formed in the last stage. The lamina transversalis anterior is formed of two separate portions. The tectum synoticum is of purely otic origin.

Notes: The fully formed chondrocranium of the Egyptian snake, Psammophis sibilans has been described in detail. The tectum synoticum is very broad and there is apparently no tectum posterius. The notochord is completely embedded in the occipital condyle. The connection between the condyle and the odontoid process persists to the fully formed stage. There is only one commissure, representing the anterior basicapsular commissure, connecting the auditory capsule to the basal plate. The fisura metotica is continuous postero-dorsally with the fissura occipitocapsularis. The distal bent end of the shaft of the columella auris represents the processus dorsalis, and the nodule that separates from it and fuses with the quadrate is the intercalare. The interorbital septum, orbitosphenoid cartilages, basitrabecular processes and planum supraseptale are completely absent. The concha nasalis is of the simplified type, the active factor in its formation is the folding of the olfactory epithelium. The solum nasi is greatly reduced and comprises the floor of the cupola anterior, the composite lamina transversalis anterior, cartilago ectochoanalis, cartilago hypochoanalis and ectochonal plate. The branchial arches are completely absent. The pterygoquadrate is represented only by the quadrate cartilage. The ceratohyals are extremely long.

Notes: The osteocranium of a late embryo of the Egyptian snake, Psammophis sibilans (age, 58 days; total body length, 190 mm) has been described. Attention was given to the relation of the different bones to the adjacent cartilages of the chondrocranium as well as to the topographical relation of the bones to each other in the late embryonic state. Several adjacent bones have not yet come in contact with each other. The membrane-bones make their appearance shortly before any sign of ossification in the cartilaginous cranium. The maxillary, palatine and pterygoid premordia have a slight earlier appearance than the other bony premordia. The lacrymal, postfrontal, squamosal, jugal, quadratojugal and epipterygoid bones are completely absent. The laterosphenoid bone is never performed in cartilage. The whole columella auris is ossified.

Notes: The morphology of the entoproct Barentsia gracilis (M. Sars, 1835), has been studied primarily by routine light microscopy techniques supplemented with observations by electron microscopy. In general, the B. gracilis adult is similar in structure to other members of the Pedicellinidae. Differences noted include: the presence of conspicuous fibers, probably collagenous, which appear to reinforce the stalk- calyx connection, the presence of a distinct glandular- appearing area (also present in the larva) of unknown function, and the presence of two rows of latero- frontal cilia on the tentacles. The first detailed morphology of an entoproct larva is also presented and the striking similarity to the adult noted. Due to the confused state of Barentsia systematics, it was found that B. gracilis, B. geniculata, and B. benedeni may very well be the same animal. This is due to the great variability, apparently due to ecological factors, found in the key taxonomic characters such as stalk morphology. The feeding behavior of the adult is described and the larval feeding and rejection mechanism is reported for the first time. The larva, while still in the brood pouch, uses its ciliary girdle to remove food from the parent's food groove. The larval feeding current is generally similar to that of the adult, with modifications which are related to the lack of tentacles in the larva. The larva differs from the adult in having special ciliated rejection pathways for the removal of excess food particles.

Notes: The cephalogenesis of chick embryos was studied at stages 3 through 11 inclusive. Ten embryos were examined at each stage. Particular attention was paid to the time of appearance of the prechordal plate mesoderm and cephalic notochord. It was found that the presumptive neural ectoderm begins to differentiate to form medullary plate as soon as it becomes associated with the chordamesoderm. Histological studies revealed the similarity of the notochord underlying the presumptive forebrain and midbrain regions, and the differences between this notochord and that associated with the hindbrain. These differences included the notochord histology and time of separation of the notochord from entoderm and neural ectoderm.

Notes: Electron microscopy of membranous labyrinths in Anolis carolinensis, Gonatodes biteanatus and Ameiva chrysolaema reveals the presences of cells of complex structure located adjacent to sensory areas in all ampullae. Microvillous cells are characterized by extensive infolding of basal cell membrane, with clefts extending to level of the basal portion of the nucleus. Each compartment thus formed is filled with mitochondria, ribosomes and glycogen. Lateral and basolateral interdigitations with surrounding cells are present. Supranuclear cytoplasm is composed of numerous mitochondria, extensive Golgi apparatus and dense populations of ribosomes and glycogen. Smooth endoplasmic reticulum is the most common reticulum present, although there is scattered granular reticulum. Toward lumen of ampulla, cells are constricted and a small portion of cytoplasm extends between supporting cells to bulge into lumen. Long, slender, branching microvilli project from luminal border into endolymph.Supporting cells resemble those found in adjacent sensory areas, with undulating luminal membranes, few mitochondria, scattered (but abundant) ribosomes and polysomes, considerable endoplasmic reticulum, extensive Golgi apparatus, lateral and basolateral interdigitations with other supporting cells and microvillous cells. Also present are large vacuoles containing elongate, randomly- oriented crystalline material.General morphology of microvillous cells suggests that they are involved in ion transport and in maintenance of electrolyte balance in endolymph. The role of glycogen in this process is discussed, as is the general problem of endolymph formation.

Notes: The development of the median eminence of several species of American anurans was studied in correlation with the progress of metamorphosis. Before metamorphosis the hypothalamic arteries break up into a plexus of capillaries lying in the extensive area of contact between the anterior lobe and the floor of the hypothalamus. During prometamorphosis the neural tissue of this area is converted into the median eminence. The neural tissue becomes thicker and shorter as the capillaries on its surface sink inward. The anterior lobe separates from the neural tissue except at its anterior tip where the residual capillaries have enlarged to form the pituitary portal veins. These changes are largely but not completely accomplished by the be- beginning of metamorphic climax. No significant alterations were found in the neurosecretory cells or their fibers during metamorphosis. The morphological data are interpreted in terms of the changing level of activity of the pituitary- thyroid axis during metamorphosis and its dependence upon a thyrotropin- releasing factor from the brain.

Notes: Leiolopisma fuscum were obtained monthly between October, 1959, and March, 1960, in northeastern Australia, latitude 16°44'S. Gross as well as histological changes observed in the gonads and accessory sex structures of lizards of both sexes revealed seasonal variation in reproductive activity, which reached a peak in December and January. During March the gonads and accessory sex structures regressed in size but by October both sexes were again reproductively active.The reproductive activity of L. fuscum, and open forest form, was compared with that of L. rhomboidalis, a related inhabitant of the rainforest. Differences in the reproductive activity of the two species were attributed to differences in the habitats and in the climatic stimuli, especially annual rainfall, associated with these habitats.Leiolopisma fuscum which occurs over much of coastal northern Australia and part of southern Papua, is far more widespread than the other species. Its cyclic reproductive pattern is probably more typical of the genus. The reproductive cycle of L. rhomboidalis is probably adaptively specialized for conditions in the rainforest, where the physical environment is subject to less seasonal variation.

Notes: The secretory processes in the shell gland of laying chickens were the subject of this study. Three cell types contribute secretory material to the forming egg: ciliated and non-ciliated columnar cells of the uterine surface epithelium, and cells of tubular glands in the mucosa. The ciliated cells as well as the non-ciliated cells have microvilli, which undergo changes in form and extent during the secretory cycle. At the final stages of shell formation they resemble stereocilia. It is postulated that the microvilli of both cells are active in the production of the cuticle of the shell.The ciliated cell which has both cilia and microvilli manufactures secretory granules which arise from the Golgi complex in varying amounts throughout the egg laying cycle. Granule production reaches its greatest intensity during the early stages of shell deposition. The ciliated cell probably supplies proteinaceous material to the matrix of the forming egg shell.The non-ciliated cell has only microvilli. Secretory granules, containing an acid mucopolysaccharide, arise from the Golgi complex. Some granules are extruded into the uterine lumen where they supply the egg shell with organic matrix. Others migrate towards the supranuclear zone. Here a number of them disintegrate. This is accompanied by the formation of a large membraneless space, which is termed “vacuoloid.” Subsequently the vacuoloid regresses and during regression an extensive rough endoplasmic reticulum with numerous polyribosomes of spiral configuration appears. It is suggested that material in the vacuoloid originating from the disintegrating granules is resynthesized and utilized for the formation of secretory product.The uterine tubular gland cells have irregular, frondlike microvilli. During egg shell deposition, these microvilli form large blebs and are probably related to the elaboration of a watery, calcium-containing fluid.

Notes: Hearts of the Atlantic hagfish, Myxine glutinosa were studied with the electron microscope after prefixation in phosphate buffered glutaraldehyde or buffered formalin and subsequent postifxation in phosphate buffered osmium tetroxide. Epicardial, myocardial and endocardial layers are identified; however the hearts of Myxine lack an extensive capillary system comparable to the coronary vessels of other vertebrate heart tissues. Instead, blood is supplied to cells via an elaborate system of channels which extend between numerous trabeculae that make up the cardiac wall of this organism. Fine structural features of special interest include the presence of numerous dense granules (chromaffin granules) within myofibers and also specific granular cells which lack the contractile elements that are characteristic of both skeletal and cardiac myofibers. Another prominent feature noted includes an elaborate system of tubular invaginations within the subjacent sarcoplasm. These elements appear to be specific for the myofibers. They are continuous with the plasma membrane and project into the peripheral sarcoplasmic matrix. Crystalline inclusions are also observed in the sarcoplasm of the myofibers. These are compared with similar inclusions in other cellular components. The Golgi complex is very extensive in the myofibers of Myxine, and granules of varying sizes and densities often appear in the vicinity of the Golgi saccules. The observations suggest that the numerous vesicles around the Golgi Complex represent intermediate stages in the formation of the chromaffin granules. The structure and function of the extensive tubular invaginations are compared with the transverse tubules reported in several mammalian heart tissues.

Notes: Normally, urodele limb regeneration is nerve-dependent. Reduction in nerve-dependency has been reported for regenerating, transplanted newt limbs (Singer and Mutterperl, '63). Aneurogenic limbs can regenerate without nerves (Yntema, '59). Induction of supernumerary limbs may be obtained from aneurogenic limbs of larval Ambystoma after transplantation orthotopically to innervated larvae and with normal nerve ingrowth to the limb transplant prevented by repeated section of brachial nerves. Of the 13 (of 43) grafts with supernumeraries, nerve counts showed 11 with 0-5; 1 with 5-10; and 1 with 20+ fibers. Orthotopically grafted aneurogenic limbs allowed to become innervated showed 14 supernumeraries in 49 grafts. This supernumerary limb induction is thus not nerve-dependent.Normally, innervated larval Ambystoma limbs grafted orthotopically and heteroplastically regenerated in 17 of 37 cases after repeated section of brachial nerves. Of the 17 regenerates nerve counts showed 4 with 0-5; 5 with 5-10; 7 with 10-19; and 1 with 20+ fibers. Larval limbs heteroplastically transplanted may require very few or no nerves for regeneration.

Notes: A fine structural analysis of fetal mouse ovaries reveals the presence of intercellular bridges between developing oocytes. These bridges, which connect two or more oocytes, are most frequently seen prior to the dictyate stage of meiotic prophase. The intercellular connections are limited by a tri-laminar membrane which is continuous with the oocyte plasmalemma. A characteristic feature of all bridges is the presence of an electron-dense material on the cytoplasmic side of the limiting membrane. Since this dense material is a constant and conspicuous component of the entire bridge, identification of these connections is possible in all planes of section. In cross section, the bridges are usually cylindrical, while in longitudinal section, a variety of configurations are observed. Oocytes connected by intercellular bridges exhibit a highly developed Golgi complex which is frequently localized in the region of the cytoplasmic continuities. Vesicular elements, apparently derived from the Golgi, are routinely observed within the boundaries of the bridges. Other cytoplasmic organelles, including rough and smooth endoplasmic reticulum, free ribosomes and mitochondria, are also seen in these bridges. The presence of these vesicles and organelles within intercellular bridges suggests that these connections may provide a means for transfer of organelles and other substances from one oocyte to another. It may be, therefore, that intercellular bridges are important for the nourishment and maturation of certain selected oocytes as well as for the synchronization of meiotic events.

Notes: The lymphatic system of 40 specimens of Dasypus novemcinctus and 5 of Dasypus sexcinctus, both found in Brazil, were injected with Prussian blue suspension, neoprene latex (followed by partial corrosion). Lymphangioradiographic study was also made. The data concern the lymph nodes, the lymphatic networks, vessels and main trunks. The main features are the following: a few lymph nodes were missing (popliteal, subpubic and ischial); the superficial jugular ll.nn. predominate on the deep ones; the dorsal mediastinal ll.nn. are well developed; a mesentericocolic plexus is formed by the many anastomoses among the mesenteric and cecocolic ll.nn.; at regular intervals there is a concentration of vessels in the submucous network of the small intestine; the submucous network of the colon shows parallel lamellar lymphatic vessels; the mucous-submucous networks are usually independent from the subserous-muscular ones; there are two types of lymphatic networks alternating along the duodenum; folliculi lymphatici aggregati are absent; the lymphatic vessels run in the mesentery independently from the blood vessels; the mesenteric lymphatic vessels present many valves; the vessels from the diaphragm terminate directly in the cisterna chyli; there is a multipolarity of the abdominal lymphatic circulation, i.e., each organ has its own lymphatic tr.

Notes: Cardiac muscle fibers have been found to extend from the left auricle along the pulmonary veins as far as the hilus of the lungs in man, in dogs, and in rodents such as the guinea pig. In other rodents, however, the cardiac muscle extends into the veins of the lung itself. To study the occurrence of cardiac tissue in the pulmonary veins, the lungs of selected rodents which represent nine superfamilies have been examined. Only the guinea pig, Cavia porcellus, was limited to having cardiac muscle in the extrapulmonary veins. All of the other 47 species examined had intrapulmonary extensions of cardiac muscle to various distances along the pulmonary veins. It is apparent that this morphological feature is not a specialization among a few families of rodents, but is probably a feature common to almost all of the rodents.

Notes: The pleiotropic effects of the dachs gene (Da) upon the skeleton have been studied in alizarin stained preparations and roentgenograms in 412 skeletons of progeny (aged 31-90 days) of three possible genotypes obtained from heterozygous parents. Basically they arise as direct or indirect manifestations of retardation, including reductions in size, deficiency in numbers of skeletal units, compensatory induction of accessory units, and overgrowth of adjacent normal units. These lead to homeotic shifts or displacement in position, crowding, and fusion. Vertebral borders anterior to v. 20 are displaced forward; those posterior tend to be backward, and the initial appearances of the epiphyses are also affected. Epiphyses of the vertebrae and long bones and the carpals and tarsals as maturity indicators are precocious as they are in retarded conditions of man, and this is enhanced in heterozygotes simulating overdominance or one gene heterosis. As indicators of premature maturation they have semblance of dysheterosis. Of particular significance is the manner in which these diverse pleiotropic effects exemplify the genetic concept of overdominance, modify the species or strain gradient pattern and the similarities of the mechanism of action to processes already revealed experimentally in the laboratory by various investigators. Extension of such study by qualitative and quantitative methods opens an approach to both genetic and environmental factors affecting normal and abnormal growth which can be tested in many ways. It would be particularly effective in study of the syndrome of effects induced by radiation, hypoxia, and other teratogenic agents.

Notes: The structure of the poison gland of the cottonmouth moccasin has been studied with the electron microscope. This gland is divided functionally and structurally into a posterior and an anterior portion. In the posterior gland the columnar principal cells are considered to be responsible for the production of the secretory material, including the toxic substances in the venom. This cell is characterized by numerous dilated membrane pairs of the granular endoplasmic reticulum and by a large Golgi complex. Four other types of cells are observed within the epithelial layer of the posterior part of the gland, but their function is unknown.The majority of the cells of the anterior part of the gland are mucous-secreting goblet cells. Some possible stages in their secretory cycle are described. Granular endoplasmic reticulum is present in the basal areas of the cell. The accumulation of recognizable secretory material appears first in structures considered to be a part of the Golgi complex.

Notes: By employing histochemical techniques, the follicular atresia and problems of interstitial cells have been worked out in the ovaries of two species of snakes (Naja tripudians and Bungarus coeruleus). Large follicles with a polymorphic granulosa show more atresia from September to November. Details of morphological and histochemical changes in the yolk nucleus substance, mitochondria and lipid bodies of oocyte and granulosa have been described. During atresia the theca interna is greatly hypertrophied and is loaded with lipids consisting of first phospholipids, then phospholipids and triglycetides and finally triglycerides, cholesterol and its ester and little phospholipids. Finally the oocyte, zona pellucida, granulosa cells and most of the cells of theca interna regress and disappear, leaving behind some residual cells of theca interna. The histochemistry of normal and atretic follicle of snakes and lizards has also been compared and contrasted with that in mammals. The “interstitial cells” of snake ovary have been discussed in the light of recent researches carried out on similar cells in the mammalian ovary.

Notes: Presumptive epidermis cells of Rana pipiens gastrula (stage 11) was induced in vitro by LiCl to differentiate into melanophores. The period from the moment when the inductor was introduced to the cells to the moment of their morphological differentiation was arbitrarily divided into three stages: “A”  -  The time interval during which the cells were exposed to LiCl, “B”  -  The period of several hours immediately following the cells' contact with the inductor, and “C”  -  The period of time immediately preceding formation of pigment in the cells. During one of these three stages the cells were treated with one of the three inhibitors: actinomycin, puromycin or ethionine. Differential sensitivity of the activated cells to the inhibitor during these three stages was revealed by their subsequent differentiation. Puromycin when applied at stage “A” increased pigmentation of melanocytes. Actinomycin acting during stage “B” in some cases caused differentiation of mesenchyme-like cells instead of melanophores. Ethionine presented to the cells at stage “C” promoted differentiation of a characteristic cell type which could not be identified with any normal histological species.

Notes: Lipid accumulations in the gonadotrophs of the pituitaries of aging and castrate fowl have been demonstrated. Secretory granules and mitochondria play no part in their formation. Some lipids, perhaps all, occupy the cavities of the ER. The ER is a highly variable organelle, appearing as spherical or irregularly shaped vesicles in gonadotrophs or as lamellae in acidophiles. The Golgi complex is typical of pituitary cells in general. There are no bodies in the enlarged cavities of the complex and convincing evidence for the origin of all secretory granules within the small vesicles of the complex is lacking. Many secretory granules appear to be without membranes and to lie free in the cytoplasmic matrix. Observations on the pituitaries of broody hens confirm earlier conclusions that the broody cells appear during the incubation period and that they are small cells with large nuclei and reduced cytoplasm. The study also confirms the accumulation of secretory granules in laying hens at one side of certain cells which are interpreted to be either LH or FSH producing basophiles. Thiouracil feeding brings about some unexpected granular accumulations in the ER vesicles of thyrotrophs of the fowl pituitary. The granules may fuse to form larger structures with concentric rings. Whether the effect is direct or indirect through the thyroid is uncertain, but thiouracil feeding and thyroidectomy produce similar effects upon the pituitary. The interpretation of these changes remains to be determined.

Notes: Bundles of nerve fibers invade the early regenerating limb of the adult newt Triturus. These fibers are unmyelinated and are only partly enveloped by accompanying Schwann cells. Isolated fibers make intimate contact with mesenchymatous cells. The fine structure of such contact regions suggests possible functional nerve terminations.

Notes: The structure of the “Corneagen,” i.e., the epidermis lying beneath the cornea-lens of the lateral eyes of the adult intermolt Limulus polyphemus was studied with light and electron microscopy.This layer is composed of heavily columnar cells containing a striking number of cytoplasmic microtubules. Many of the microtubules are grouped into compact bundles or fascicles, generally each cell having at least one microtubule bundle. The cornealens end of each cell has numerous microvilli, each with a core of delicate filaments. The crypts between microvilli end in extracellular expansions and plaques of electron dense amorphous material are associated with these terminal expansions. Cytoplasmic microtubules appear to insert into these dense areas.The basal ends of the cells are thrown into many pseudopodial processes which extend into the surrounding extracellular space. The cytoplasm of the pseudopodia is composed largely of microtubules and their associated low density halos.Junctional complexes consisting of zonulae adhaerens and septate desmosomes are present between adjacent cells. Mitochondria, ER, cytoplasmic vesicles, Golgi stacks and other ultrastructural details of the epidermal cells are described. The ultrastructure of a column of pigment free processes lying between the apex of the lens cone and the underlying photoreceptive portion of the ommatidium is also described. Ducts or vessels of uncertain origin are present in the inter-ommatidial spaces.Possible roles played by the microtubules, the significance of their disposition and of their association with the dense subsurface plaques are discussed in terms of intracellular support, epidermis-lens attachment and extracellular pattern determination. In addition, the likelihood of the dense plaques being the site of microtubule assembly is considered.

Notes: Differentiating oocytes and associated follicle cells of two species of amphineurans (Mollusca) Mopalia muscosa and Chaetopleura apiculata have been studied by techniques of light and electron microscopy. In addition to the regularly occurring organelles, the ooplasm of young oocytes contains large, randomly situated, basophilic regions. These regions are not demonstrable in mature eggs.As oocytes differentiate, lipid, pigment and protein-carbohydrate yolk bodies accumulate within the ooplasm. Concomitant with the appearance of pigment and the protein carbohydrate containing yolk bodies, the saccules of the Golgi complex become filled with a dense material. Associated with the Golgi complex are cisternae of the rough endoplasmic reticulum which are filled with an electron opaque substance which is thought to be composed of protein synthesized by this organelle. That portion of the cisternae of the endoplasmic reticulum facing the Golgi complex shows evaginations. These evaginations are thought to finalize into protein containing vesicles that subsequently fuse with the Golgi complex. Thus, the Golgi complex in these oocytes might serve as a center for packaging and concentrating the protein used in the construction of the protein containing pigment or protein-carbohydrate yolk bodies. The suggestion is made that the Golgi complex may also synthesize the carbohydrate portion of the formentioned yolk bodies.In an adnuclear position in young oocytes are some acid mucopolysaccharide containing vacuolar bodies. In mature eggs, these structures are found within the peripheral ooplasm and we have referred to them as cortical granules. There is no alteration of these cortical granules during sperm activation.

Notes: Communication organs (septulae) of cheilostome Bryozoa are more complex than perviously believed. Annuli, present only in lateral septulae, are thickenings of the intercalary cuticle. Each communication pore is filled with a ring-like “pore cincture,” through which project a pair of “special cells.” Septulae of all species examined (10 species from 6 families) can be considered modifications of the same structure, varying only in degree of calcification and number of communication pores.External walls, including basal and lateral walls, are best defined as reinforcements of the ectocyst, which is derived by intussusception from the primary cuticle of the ancestrula. The lateral ectocyst must be considered a double layer formed by invagination of the distal ectocyst. Internal walls are developed by apposition from inner parts of the ectocyst; they include pore plates and transverse walls.External walls are laid down first. Lenticular masses develop unilaterally on the uncalcified lateral ectocyst; the pore plate develops by apposition from the interior part of the ectocyst. Depending on the species, the pore plate may or may not be calcified at the time of its formation. Communication pores are formed when the developing pore plate abuts against embryonic special cells. The septular ectocyst never calcifies; it breaks down when the pore plate is complete.Some ascophorans undergo “reparative budding,” in which new zoids are formed within dead zoecia. Hollow, ectocyst-covered buds lined with blastemic epithelia are produced from septulae of live zoids; adjacent buds may fuse.These findings are consistent with the view that lateral septulae are aborted zoids and that pore plates represent transverse walls.

Notes: The development of the motor horn of the foetal mouse was investigated. A sampling technique was evolved using cresyl violet stained material.A decline in the number of motor neuroblasts occurred during the development, on each side, of four definitive motor regions from a single longitudinal column. The total number of motor horn cells fell from about 100,000 on the eleventh day after mating to about 25,000 on the fourteenth day. The early stages of this decline (between 11 and 12 days) are probably explained by the fact that not all neuroblasts in the region differentiate into motor cells; he later decrease can be entirely accounted for by the number of degenerations.Irradiation of the foetal mouse with a dose of 50 rads of cobalt-60 gamma-radiation resulted in an excess of about 20,000 differentiating motor cells on the thirteenth and fourteenth days. Irradiation increased the actual number, but not the percentage, of degenerations. This suggests that differentiation is a phase in the growth process which proceeds degeneation.

Notes: Histochemical procedures for acid phosphatase in normal and lens-regenerating eyes of the urodele Diemictylus viridescens demonstrate activity in a variety of structures. In the normal urodele eye, acid phosphatase is present in conjunctival and corneal epithelial cells and associated glands, in blood vessel endothelium and posterior epithelial cells of the iris, in the anterior lens epithelium, and in the cytoplasm of the optic nerve. Acid phosphatase in the lens-regenerating eye is localized in the same structures as in the normal eye as well as in increased amounts in the corneal epithelial cells and stromal macrophages at the lentectomy wound site and in the posterior portion of the developing lens during completion of differentiation of primary into mature lens fibers characterized by loss of many intracellular organelles. On the basis of these histochemical findings, it is proposed that hydrolytic lysosomal enzymes play an important role in the processes of cellular and intracellular destruction and synthesis which occur during Wolffian lens regeneration in the urodele.

Notes: This investigation is a histological study of the development of the ovary of the sea lamprey from south-western Ontario, Canada. Development is described from the first appearance of primordial oögonia at the site of the ovary up to ovulation. After mitotic proliferation, oögonia enter meiotic prophase and become oöcytes which enlarge and acquire basophilic cytoplasm.During metamorphosis all oöcytes are at a uniform stage of development and no oögonia remain in the ovary.The follicular layer in the early adult produces nurse cells which are incorporated into the oöcyte and increase its mass of RNA-rich cytoplasm. As the oöcyte enlarges, proteid yolk platelets are laid down in the cytoplasm. Vacuolation of the nucleolus, indicative of protein synthesis, is extensive during period of rapid growth and yolk formation.Immediately prior to spawning there is an accumulation of fluid under the follicular layer and the oöcyte emerges through a mound of follicular cells.Atresia of oöcytes occurs throughout the adult stages and following spawning. It is characterized by an enzymic dissolution of the yolk followed by phagocytic invasion. The phagocytes arise from the follicle in the early adult stages and from an unidentified source in the spawning-phase adult.

Notes: Frog retinal rod outer segments were detached from dark adapted retinas by (1) agitation in frog Ringer's solution or (2) by crushing between two glass surfaces. The resulting suspensions were further purified by low and high speed centrifugation procedures in Ficoll density gradients. The density of the outer segments in Ficoll solutions was found to be 1.09. The large frog outer segments, unlike bovine outer segments, are not readily separated from nuclei, which were estimated to comprise 2.6-8% of the material, based on DNA analyses. The RNA/DNA ratio was 0.4-0.5, like that of neuronal nuclei. Representative enzymes of glycolysis (lactic dehydrogenase and glyceraldehyde phosphate dehydrogenase), phosphogluconate oxidation (glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase), the citric acid cycle (malic dehydrogenase) and ATP degradation (ATPase) were assayed. A major part of the malic dehydrogenase activity was probably due to inner segments attached to some of the outer segments. Glyceraldehyde phosphate dehydrogenase and glucose-6-phosphate dehydrogenase (but not lactic dehydrogenase) activities were lower in detached outer segments purified on Ficoll gradients than in samples of outer segment layer microdissected from freeze-dried sections of frog retina, as well as in whole retina. The data suggest that the activities of all the enzymes studied are intrinsically low in rod outer segments.

Notes: The frog, Rana pipiens, hibernates through the winter with ovaries containing oocytes which, in size and appearance, are ready for ovulation and maturation. From November through April, the normal time of egg laying, ovulation and maturation can be induced by interrupting hibernation and administering gonadotropic hormones. In the studies reported here, it has been found that oocytes taken from hibernating animals in early winter take up amino acids from a saline medium at a relatively rapid rate. Respiratory inhibition produced by such agents as dinitrophenol (DNP) and anaerobiosis, does not completely stop uptake but slows it down markedly.In late winter, amino acid uptake tends to be slower in normal cells and when DNP or cyanide is added, a marked acceleration of the rate of uptake is observed. The uptake in poisoned cells is accumulative, producing internal concentrations higher than that of the medium. At this concentration of DNP, amino acid incorporation is almost completely stopped. Fluoride abolishes the DNP stimulation of amino acid uptake. Removal of sodium ion from the incubating medium has no effect on either uptake or incorporation.These data are interpreted to mean that a capacity for oxidative phosphorylation in oocytes diminishes during winter hibernation. In the spring either an anaerobic capacity comes into being or becomes capable of being switched on. During this time the rate of amino acid uptake, even in the downhill phase, is limited by energy availability and is not dependent on a sodium gradient.

Notes: The effect of temperature on intracellular ATP levels in cells of the ascitic Hepatoma 129 has been determined using an UV-enzymic method. ATP levels are found to vary inversely with temperature over the range (4°-37°C) studied, and this variation is found to be reversible. An indication of a possible direct correlation of ADP level with temperature was also obtained.A correlation is deduced between rise in intracellular ATP level and decrease in cytoplasmic viscosity, both from the present results, and from similar effects recently reported by Landau ('61) for increase in pressure.The significance of these observations is discussed with reference to the model for cytoplasmic viscosity proposed by Marsland ('56) and the wider biological significance of this effect is explored.

Notes: Analysis of the equilibrium of the normal biconcave human red cell, in terms of its tension and a pressure inside, suggests a force of attraction between the opposite membranes at the dimple regions. The analogous attraction that causes rouleaux formation is mediated by long-chain molecules. Single cells hanging on edge between polarizer and analyser, almost “crossed,” were photographed at different angles to the axis of the polarizer. Enlarged prints were scanned by a photometer. For single cells the records showed non-significant fluctuations of intensity, but mean values for 32 cells showed a very significant sinusoidal variation with angle, as predicted by theory for birefringence in the cell at the dimple region. For the rim region, the averaged data showed no variation with angle. In cells moderately osmotically swollen, birefringence in the centre of the dimple region was absent, but persisted close to the inside of the membranes. The latter disappeared in cells further swollen to a biconvex shape. The data is interpreted as indicating oriented chains of molecules across the interior of the cell at the dimple region. The behaviour on swelling was what had been seen in a model with nylon fibres oriented between the charged plates of a condenser, in which the variation of attractive force with distance was adequate to explain the equilibrium of the red cell membrane.

Notes: H3-Uridine microinjected in the giant axons of the squid is incorporated in a TCA insoluble material. There is no difference between stimulated and resting axons as to the amount incorporated.The amount incorporated is increased if the stimulation precedes the microinjection of the tracer.RNA was purified and characterized from the axoplasm, axon sheaths and from a purified membrane preparation obtained from squid retinal nerve.

Notes: Diploid human fibroblasts in culture (WI 38) were allowed to reach a stationary phase and were then stimulated to reenter DNA synthesis and cell division by addition of serum to the culture medium. The rate of protein synthesis increased during the first hours after addition of serum reaching at three hours a plateau value that continued for at least 24 hours after serum addition. Inhibition of protein synthesis during the early hours after serum addition abolished the stimulation of DNA synthesis occurring 20 to 28 hours later.Increased protein synthesis was preceded by a rapid decrease in the intracellular pool size of most amino acids. These changes were independent of concomitant protein synthesis. They suggest that serum exerts an immediate effect on the function of the cell membrane.

Notes: Six diploid human fibroblast strains were grown in confluent monolayers. Holes were scraped in these monolayers and the number of cells proliferating into these “wounds” with time were determined. The migration and mitotic aspects of the proliferation of fibroblasts into these wounds were analyzed separately.Small amounts of undialysed or dialysed serum were essential for cell division but not migration. Saline extracts of skin could not substitute for serum in the medium. Neither zinc nor cupric ion at tolerable concentrations (10-5M) increased the rate of cell proliferation.Normal human fibroblasts did not immediately start to divide from confluency into the “wound” space. Their generation time was about 32-39 hours. Fibroblasts from patients with cystic fibrosis began to divide almost immediately into the “wounded” area. Their generation time was about 48 to 56 hours.

Notes: Ribosomal RNA synthesis was selectively inhibited in HeLa cells by lucanthone, a clinically useful schistosomicide which shares many of the properties of Actinomycin D. Synthesis of DNA-like RNA continued during complete inhibition of ribosomal RNA synthesis. Under these conditions newly synthesized DNA-like RNA accumulated normally in polyribosomes of the cell cytoplasm; most of it appeared to be messenger RNA. DNA synthesis was partially inhibited by lucanthone but protein synthesis was undisturbed. Synthesis of ribosomal RNA promptly resumed after removal of lucanthone and cell survival was not affected if exposures to the drug were limited to two hours.

Notes: Infection of Escherichia coli: with T4 bacteriophage causes the appearance of a new valyl-tRNA synthetase activity associated with a molecule that, compared to the host enzyme, exhibits a greater resistance to denaturation by heat or urea, a larger molecular volume, a higher rate of sedimentation in sucrose gradients, a greater net positive charge, and a greater ability to charge yeast tRNA. No evidence has been found for similar changes in synthetase activity for the other amino acids. Appearance of the new activity requires continued protein synthesis and results from a modification of the preexisting host enzyme rather than de novo synthesis of a totally new enzyme. By 20 minutes after infection at 30°C, all of the host enzyme has been converted into the new form. Phage mutants have been isolated that fail to effect a normal conversion. The properties of these mutants suggest that conversion involves the addition, to the host enzyme, of a protein specified by the phage genome. Drastic reduction of phage-induced activity in one of these mutants does not interfere detectably with phage development in a normal host, suggesting that the presence of the new activity is not essential for normal phage production.

Notes: A Qβ RNA segment was produced in vitro by means of a synchronized minus-strand-directed synthesis using purified Qβ replicase, a reaction known to yield infectious Qβ RNA. Arguments are presented that a 5.5S product obtained by incubation for 30 seconds at 20°C corresponds to the 5′-terminal region of Qβ RNA. After obtaining and sequencing 26 T1 and 21 pancreatic RNase products, we established 3′ nearest neighbors by using products labeled with only one nucleoside [α-32P]triphosphate at a time. The ordering of the oligonucleotides was facilitated by observing the time of their appearance in products from incubations of 5, 10, 15, 20, and 25 seconds. The enzymatic product consists of 160 nucleotides; no known initiation triplet appears before the sixtieth nucleotide. There is no indication that the coat protein cistron starts within the RNA segment analyzed.

Notes: DNA-dependent RNA polymerase reacts with a nucleic acid to form a binary complex, which can be isolated by density gradient centrifugation or by millipore filtration techniques. Studies of the complex of Escherichia coli RNA polymerase and phage lambda DNA show that temperature and ribonucloside triphosphates do not affect the extent of binding. The binary complex is very sensitive to ionic strength. Enzyme bound in the complex exchanges with free enzyme. In the presence of Mg2+, the lambda DNA becomes saturated with enzyme when 2.5 μg have been bound per microgram of DNA. Transfer RNA and synthetic polyribonucleotides also react with the enzyme at 0°C. In the presence of divalent cation at low ionic strength, the main complex of the enzyme and a polyribonucleotide has a sedimentation value of 15S. In the absence of divalent cation, or at higher ionic strength, the value is 13S. Polyuridylate and polycytidylate have a greater affinity for the enzyme than polyadenylate or transfer RNA. An interesting question regarding the specificity of interaction of RNA polymerase with native DNA is whether any of the RNA chains made have the sequence AUG, the principal N-formylmethionine codon, at the 5′ termini. Studies with T7 DNA show that, under conditions of high ionic strength, 24% of the RNA chains start August.

Notes: A cell line was established in vitro from a spontaneous myeloid leukemia of SL strain mice. This cell line was cytologically identified as myeloblast, and its normal differentiation appeared to be completely blocked in mass culture. When the line cells were seeded in soft agar with a conditioned medium from normal cells, either macrophages or neutrophil granulocytes appeared from a single clone. The rate of formation of colonies containing differentiated cells always increased with an increase in the concentration of conditioned medium. The conditioned medium from this line cell was not as effective as was that from normal cells in inducing differentiation.

Notes: Considerable variability has been found in the yield of cells in batch cultures of Tetrahymena pyriformis grown axenically in 1% tryptone/0.05% yeast extract. This variability has been traced to the photolysis by visible light of the flavin mononucleotide and thiamine components of yeast extract.

Notes: Colony formation in vitro by mouse bone marrow cells following stimulation by human urine was analysed over a 7-day incubation period. There was a linear increase with time in the number of cell aggregates (clusters) developing in such plates. Early in the incubation period all clusters were granulocytic although later macrophage clusters developed. Although most fully developed colonies were composed of macrophages, mapping and transfer studies showed that at least half of these had initially arisen early in the incubation period as granulocytic clusters.

Notes: Maturation of bacterial viruses requires the formation of mature viral nucleic acid in the form found in progeny virus particles, the “packaging” of this viral nucleic acid within a capsule, and the assembly of “packaged” nucleic acid with the accessory structures necessary for infectivity. Recognition that the replicating viral nucleic acid is frequently in a form distinct from that found in mature particles has, in some instances, led to the postulation and formal description of presently unknown processes that must participate in the synthesis of the mature form. In at least some instances, packaging of the mature nucleic acid is clearly integrated with its synthesis.Examples involving the DNA bacteriophages T4, λ, and φ X174 and the RNA bacteriophages are presented to illustrate these points.

Notes: The development of a virus is programmed by a series of negative and positive controls which determine the timing and the segment on either of the two DNA strands (l or r) to be transcribed into specific messenger RNA's. Bacteriophage λ provides one of the most deeply studied systems for following the development of lysogenic viruses. In the lysogenic repressed state, only 2-4% of the λ genome is expressed. This pc-cI-rex region is transcribed leftward to produce a repressor protein which prevents any further transcription by blocking the oL and oR operators flanking the cI-rex operon (figs. 1, 2). This negative control is relieved by destruction of the repressor, and the result is the “induction” of viral development. The earliest post-induction or postinfection events are the leftward transcription of the pLoL N region from strand l and the rightward transcription mainly of the pRoR-x segment from strand r. The N product acts as a positive control, permitting a leftward transcription beyond gene N and a rightward transcription of genes cII-O-P and also Q. The int-xis system controls the excision of the λ genome, whereas the act of rightward transcription and the products of genes O and P initiate the replication of λ DNA. The product of gene Q, still another positive control, stimulates rightward transcription of the late genes which control the synthesis and assembly of the phage heads and tails as well as cell lysis. Among other types of negative control are the possible competition between the two divergent transcriptions originating in region x, the “antirepressor” effect of the x product, and the interference between the two convergent transcriptions which collide in the central b2 region. The majority of controls are based on protein-DNA interactions and can be modified by mutations. For instance, transcription can be rendered independent of negative repressor control either by constitutive, v, mutations which decrease or abolish the affinity of the o operators for the repressor or by insertion of new promoters-e.g., c17 or ric- on the “downstream” side of the operator. The need for the positive N and Q controls may also be obviated by mutations in the N- or Q-dependent promoter or terminator elements.The specific DNA structure within the controlling sites is not known. However, a remarkable coincidence was observed; namely, the occurrence of pyrimidine-rich clusters in those segments of the individual DNA strands acting as templates for RNA synthesis. This observation, which pertains to all studied DNA's, including those of phages T2, T3, T4, T5, T6, T7, λ, and φ 80, formed the basis for a proposal that implicates pyrimidine-rich clusters in the initiation, control and/or termination of transcription, and also in the determination of the preferred strand and, consequently, the orientation of transcription. General considerations regarding the possible role of the structural singularities, especially those represented by the pyrimidine clusters, in the bipartite structure of the recognition regions in DNA are discussed.

Notes: After the experimental verification of Crick's adaptor hypothesis for the role of tRNA, it became apparent that one of the most important of the protein-nucleic acid interactions occurs at the first step in protein synthesis, namely the amino acid activation reaction. It is here that a specific aminoacyl-tRNA synthetase must select, with high fidelity, a specific tRNA out of a large collection of molecules of similar size, shape, and overall composition. A mistake at this point, either by esterification of the wrong amino acid to the correct tRNA or by selection of the wrong tRNA, will inevitabley result in the insertion of an amino acid at an incorrect position in a growing polypeptide. Although there are known rules that dictate how one nucleic acid can recognize and interact with another nucleic acid, nothing is known regarding the mechanism by which a specific protein can recognize and interact with a specific nucleic acid. In order to gain some insight into the specific recognition between an aminoacyl-tRNA synthetase and its cognate tRNA, it became necessary to study the specific interaction with highly purified materials, preferably in gram quantities. An effort to do this for both the synthetases and the tRNA's was launched at the Oak Ridge National Laboratory about 6 years ago. Four high-resolution column chromatographic procedures have been developed in the ORNL Macromolecular Separations Program for the separation and production of highly purified species of tRNA's. An unexpected “spin-off” from this program is the analytical use of some of these systems to detect qualitative changes in the tRNA profile of cells as a consequence of virus infection, methionine starvation, and other metabolic alterations.Some examples of the heterologous interaction between aminoacyl-tRNA synthetases of one species with the tRNA's of another species, and some of the inherent dangers in the interpretation of such interactions, are considered.Finally, some speculations are made regarding the possible role of tRNA in regulation.

Notes: Several steps in the synthesis in vitro of infectious bacteriophage RNA can now be described. The reaction catalyzed by the Qβ RNA polymerase is known to involve several components, including the enzyme, host cell factors, Qβ RNA template, and the strand complementary to the Qβ RNA. The interaction of these components and the mechanims of the reaction appears to be considerably more complex than was proposed in earlier models.

Notes: Theoretical osmotic fragility curves were calculated and drawn by computer using the van't Hoff equation and the isotonic areas and volumes of 1000 individual erythrocytes. We studied the influence on the calculated curves of theoretically altering the fraction of the volume which was osmotically active from 50 to 70%, and of altering the permissible stretch before hemolysis from zero to 10%. With the two assumptions-that the membrane does not stretch before hemolysis, and that the osmotically active fraction of the cell volume is 0.58-it was possible to duplicate the general shape of the standard fragility curve; the exact NaCl concentration, however, at which there was 50% hemolysis was approximately 0.1 gm/100 ml higher than found in vitro. The calculated osmotic fragility curves can be made quantitatively similar to in vitro ones if the following statements are true: the osmotically active volume is 58%, the permissible stretch of the membrane without lysis is 6%, the cell membrane resists a slight osmotic pressure gradient of approximately 0.1 atmospheres, and hemolysis is an all or nothing phenomenon. This set of values for the relevant factors is sufficient but not unique in causing the superposition of the calculated and experimental curves. The frequency distribution of the cells according to the hemolytic salt concentrations (the sodium chloride concentration at which an individual cell just hemolyzes) was skewed positively and was leptokurtic for each of the seven normal subjects studied.

Notes: The fraction of cell capable of division was determined for (1) population of the human diploid cell strains, WI38 after different numbers of subcultivations in vitro and (2) a single population of WI38 cells at intervals throughout its entire in vitro lifespan. In both cases the percentage of cells capable of division decreased with increasing age in tissue culture. The rate and the magnitude of the decrease is sufficient to account for the limited in vitro lifespan reported by other investigators. Furthermore, the decrease in the fraction of cells capable of division in similar in some respects of senescence among human populations.

Notes: A method is described which maintains viable erythroid cells in tissue culture for periods from nine to twenty days. These cells appear predominantly as small round cells with scanty cytoplasm. They synthesize both heme and globin and are relatively more numerous free in suspension than in the adherent monolayer. Ferritin isomorph may serve as a convenient marker in tissue culture of cells of erythroid origin, suggesting that such cells may persist despite a completely transformed appearance and a loss of the ability to produce hemoglobin.

Notes: The conformation of native double helical DNA is well-known, but it is possible that small regions occur within native DNA, undetectable by X-ray diffraction methods, which have different conformations. Model structures are the synthetic deoxypolynucleotides of defined sequence. Under the conditions used, DNA, poly d(A-T) • poly d(A-T), and poly d(T-G) • poly d(C-A) can all give similar X-ray diffraction patterns, whereas poly dA • poly dT, poly dI • poly dC, poly dG • poly dC, and poly d(T-C) • poly d(G-A) clearly differ from DNA. This led to the tentative hypothesis that those DNA's in which all purines are in one strand and all pyrimidines in the other differ in structure from those (such as native DNA) in which purines and pyrimidines alternate or are irregular. We now find that poly d(I-C) • poly d(I-C) does not fit the hypothesis and is a most unusual structure, having seven or eight base pairs per turn. Both molecular model building and circular dichroism studies suggest that it is a left-handed helix. A number of purified tRNA's have been crystallized. We have obtained, from unfractionated tRNA, crystalline “powder” X-ray diffraction patterns showing rings and spots to about 20 Å resolution. It is not clear whether cocrystallization has occurred, or whether there is fractional crystallization, though preliminary evidence favors the latter. Determination of the structure of crystalline tRNA has many features in common with protein crystallography, but there are a number of distinct differences.

Notes: All tRNA sequences so far known can be folded into a cloverleaf structure. Physical data and chemical reactions allow us to draw conclusions on secondary (cloverleaf) and tertiary structure. N-oxidation of adenosine to adenosine-1-N-oxide can be done with monoperphthalic acid in non-base-paired regions of polynucleotides and can be followed easily by changes in absorption of ultraviolet light. Thus this method can be used to determine the structure of tRNA's. A fingerprint of the N-oxidation product of tRNAyeastPhe reveals that all adenosine residues are protected except the 3′-terminal adenosine and the three adenosine residues in or adjacent to the anticodon. On this basis a conformation of tRNAyeastPhe is proposed. Similar tertiary structures can be constructed for the other tRNA's. In order to connect tertiary structure of a tRNA and recognition by its aminoacylating enzyme, the rate of aminoacylation, as a function of temperature, was measured. Neither changes in the anticodon nor specific changes at the 3′-terminal adenosine abolish aminoacylation. Single crystals of tRNAyeastPhe were obtained from aqueous solutions upon addition of various organic solvents.

Notes: Assemblies of protein molecules represent a fundamental level of biological organization. The dynamic behavior of these systems-including both the assembly process and functional rearrangements-may be accounted for by the specificity of the protein interactions, which depend on environmental conditions. Analysis of the self-assembly of virus particles has established that the design of an ordered structure can be built into the specific bonding properties of the constituent proteins. Any structure which can change its state of organization is, by definition, polymorphic. The distinctive aspect of polymorphism in protein structures, contrasted with nonliving states of matter, is that the molecular design has been selected to carry out a function and that this function is part of an integrated system. The differences in molecular conformation and arrangement in all polymorphic structures-for example, allosteric enzymes or ice crystals-depend on the intrinsic interaction properties of the molecules themselves. The structures of ice and water illustrate relations between specificity and polymorphism which are relevant to the form and function of protein assemblies.Two types of polymorphism can be distinguished: modal polymorphism, which is externally moderated, as in phase transitions between different crystals forms; and positional polymorphism, which is internally moderated, as in the different disposition of identical molecules within a single crystal lattice. Positional polymorphism, exemplified by the quasi-equivalent bonding of icosahedral virus coat proteins and the different arrangement of myosin and paramyosin at the center and polar portions of the bipolar filaments, results from specific interactions that are not compatible with a strictly equivalent packing of identical molecules. The structural rearrangements in muscle contraction and the switching between the oxy and deoxy forms of hemoglobin represent the formation of different structures in response to altered external conditions. The different structural states of many protein assemblies are characterized by conserved connections which may be regarded as providing the framework for functional rearrangements. The types of polymorphism displayed by hemoglobin, virus, and muscle proteins demonstrate the relevance of the simple view that the function of a protein is determined by the potential structures it can form.

Notes: Water exchange has been studied in ovarian and body cavity eggs of three Anuran species (Rana esculenta, R. pipiens and R. temporaria) with the isotope exchange method using the automatically recording diver balance. In order to estimate the rate of water diffusion in egg cytoplasm (D), a factor required to determine the rate of water permeation (= the exchange coefficient, E), three different chemical treatments (digitonin, ethanol and formaldehyde) have been used to remove the surface barrier to water flow. The obtained mean value of D, 5 × 10-6 cm2 sec-1, has been accepted as a close approximation of water diffusion in the egg cytoplasm. This value has been used to determine the exchange coefficients in the egg types of the above mentioned species. Comparisons have been made between the calculated values of E both within and between species, and some suggestions have been made concerning the influence of microvilli on the rate of water exchange. Although, on the average, water exchange proceeds at a greater rate in ovarian than in body cavity eggs, a measurable diffusion barrier at the surface can be demonstrated.

Notes: Cellular transformation was induced with avian myeloblastosis virus strain BAI-A (standard AMV) and with a strain of AMV containing subgroup B only. Cultures of muscle tissues from either chick embryo or day old chicks were used for this study. Results were similar in C/O and C/A cells. Leukemogenic virus was continuously produced by these transformed cultures.

Notes: In the presence of 1 mM ATP in the external medium, TA3 mouse ascites tumor cells showed a dramatic loss of potassium and gain of sodium down their respective concentration gradients. The volume changes detected by size discrimination with the Coulter counter have been adequately confirmed by densimetric techniques. Further, some experiments were so designed that net losses of both ions occurred and the cell shrank in response to ATP, a response which was predictable if the volume change was produced by a loss of cell water. We believe that ATP produces a major change in the passive permeability of the membrane to these ions and the effect may be due to a response of a contractile protein in the membrane to ATP.

Notes: Growth hormone, oxytocin, parathyroid hormone, prolactin and lysine vasopressin strongly stimulate mitotic activity in rat thymocyte populations maintained in vitro. These hormones have no mitotic effect on cells maintained in calcium-free medium. It is concluded that they stimulate mitosis only indirectly by sensitising the mitotically competent segment of a thymocyte population to the action of calcium. The stimulatory action of calcium itself is opposed by low concentrations of the mucopolysaccharide chondroitin sulphate. However, the inhibitory action of chondroitin sulphate can be overcome by growth hormone.A possible common mechanism of action of these hormones on mitotically competent cells is discussed.

Notes: A system for fractionating populations of living cells by velocity sedimentation in the earth's gravitational field is described. The cells start in a thin band near the top of a shallow gradient of 3% to 30% fetal calf serum in phosphate buffered saline at 4°C. Cell separation takes place primarily on the basis of size and is approximately independent of cell shape. A sharply-defined upper limit, called the streaming limit, exists for the cell concentration in the starting band beyond which useful cell separations cannot be achieved. This limit, which varies with the type of cell being sedimented, can be significantly increased by proper choice of gradient shape. For sheep erythrocytes (sedimentation velocity of 1.6 mm/hour) it is 1.5 × 107 cells/ml. Measured and calculated sedimentation velocities for sheep erythrocytes are shown to be in agreement. The technique is applied to a suspension of mouse spleen cells and it is shown, using an electronic cell counter and pulse height analyzer, that cells are fractionated according to size across the gradient such that the sedimentation velocity (in mm/hour) approximately equals r2/4 where r is the cell radius in microns. Since cells of differing function also often differ in size, the system appears to have useful biological applications.

Notes: The progression of rat thynocytes (maintained in vitro) into mitosis is profoundly affected by the level of magnesium in the medium. Increasing the extracellular MgC12 concentration from 0 to 1.0 mM does not affect mitotic activity, but a further increase to 1.2 and 2.5 mM rapidly and strongly stimulates the flow of cells into mitosis. The level of extracellular magnesium also governs the mitogenic effectiveness of calcium ions. The mitogenic action of magnesium stems from an ability to stimulate the initiation of deoxyribonucleic acid synthesis.

Notes: Colony-forming cells (CFU), which have the general properties of hemopoietic “stem” cells, appear to be augmented in the mouse fetal liver from 12-18 days gestation and then decrease in the newborn. This finding suggests that few, if any, hemopoietic “stem” cells remain in the adult liver, an organ which appears to be unable to function erythropoietically, even at times of severe crises. In the spleen, and active adult as well as embryonic hematopoietic organ, the total number of CFU increases from 18 days gestation until at least 7 days after birth.Spleen and liver CFU augmentation seems to occur in cojunction with an analogous expansion of non-hematopoietic cells. The data suggests, in fact, that while there is an increase in the total number of liver CFU, there is also a dilution of liver CFU in the total cell population at successively later gestational ages.

Notes: The incorporation of tritiated thymidine and deoxycytidine into DNA of x-irradiated mammalian cells was studied. Both inhibition and stimulation were found due to pool changes rather than to effects on DNA synthesis, indicating that precursor uptake can be a misleading method to measure DNA synthesis rate.

Notes: A series of aldehydes of varying protein crosslinking strengths have been tested on intact and internally perfused crayfish axons. Non-crosslinking aldehydes have no effect, or cause a gradual decline in resting potential and overshoot with no widening of the spike. Strong crosslinking compounds, such as acrolein, crotonaldehyde, and glutaraldehyde, widen the action potential significantly while reducing its amplitude. Differences in the shapes of the resulting action potentials and accompanying impedance changes suggest that each crosslinking aldehyde exerts different effects on the axon. Glutaraldehyde, the strongest crosslinking agent tested, slows both rising and falling phases of the spike, and also of the impedance change, suggesting a prolongation of the transient increase in sodium conductance. The ability of protein crosslinking agents to alter excitability, and particularly to slow the various phases of the action potential, provides support for the hypothesis that a conformational change in a protein or protein-phospholipid complex is involved in excitation.

Notes: Growth of KB cells was inhibited by both spermine and spermidine, but the inhibition is reduced in conditioned medium. The amount of spermine required for 50% inhibition of plating varied according to the type of serum used with medium 199 (calf, fetal bovine, and horse; 0.55, 0.9, and 24 μg/ml respectively). The spermine oxidase activity of the three sera was calf 〉 horse 〉 fetal bovine, which is not the same ordering as was obtained for the inhibition. When the concentration of sera in the media was varied, the inhibition decreased as calf and fetal bovine sera concentration increased, whereas, with horse serum, an increase in serum concentration increased the inhibition. The opposite effects of increasing concentrations of the sera on the inhibition suggest that at least two factors are involved in the inhibition. A scheme which involves three factors (spermine oxidase, another enzyme and its activator) is postulated to account for the inhibitions and reversals observed. Spermine oxidase alone cannot account for the action of polyamine on cells.

Notes: Reduction in alkaline phosphatase activity was observed when HeLa S3 cells were grown in Puck's medium containing high concentrations of human serum. This effect was not seen with the enzyme of Chang liver 8A cells. The induction of increased alkaline phosphatase in HeLa S3 by prednisolone or by osmolality changes was not prevented by serum. The concentration of serum in the culture medium had no influence on acid phosphatase activity.

Notes: These studies have been conducted as a first step toward investigating the molecular mechanism by which DNA-dependent RNA polymerase recognizes a specific region of DNA. The DNA moieties are either bound to or freed from RNA polymerase. Results obtained so far indicate that the DNA moieties remaining as resistant segments after deoxyribonuclease digestion should be regarded as the binding regions on DNA. The results described here were carried out by use of E. coli and lambda DNA.

Notes: In cultures of differentiating chicken embryo muscle cells there is a steep decline in DNA polymerase activity which closely parallels the time of rapid cell fusion and the formation of multinucleated myotubes. The DNA polymerase activity remaining in the cultures is almost completely associated with single unfused cells. Cell fusion does not require a confluent culture and fusion capability appears to be severely reduced in the remaining single cells following an approximately ten hour time period during which the majority of fusion takes place. A model is presented to explain the observed kinetics of cell growth and cell fusion in vitro.

Notes: Synchronous cultures of HeLa cells obtained by selective detachment of mitoses were treated with high concentrations of thymidine. The inhibitor was added soon after completion of cell division and rates of cell enlargement and accumulation of DNA, RNA and protein were compared for untreated and thymidine-treated cultures at various points of the cell cycle. It was found that concentrations of thymidine which in randomly growing cultures inhibit the rate of cell division by more than 90% allowed a considerable degree of DNA synthesis and did not affect the rate of accumulation of RNA and protein, when applied to cells in the G1 phase of synchronous culture. Treated and untreated cells enlarged at the same rate throughout their life cycle. The results show that concentrations of thymidine commonly employed to produce cell synchrony do not arrest the cells at the G1-S boundary, but allow slow progress through S in respect to DNA synthesis, and near-normal progress towards G2 as regards RNA and protein accumulation and cell enlargement.