Showing papers in "Journal of Cell Science in 1969"

On the surface coat and flagellar adhesion in trypanosomes.

Abstract: Pathogenic trypanosomes in their bloodstream phase have a smooth and compact coat 12-15 nm thick enveloping the entire surface membrane of the body and flagellum. In the sleeping-sickness trypanosome Trypanosoma rhodesiense this coat is absent from the stages of development in the midgut of the tsetse-fly vector and from their counterparts obtained by cultivation of the trypanosome in vitro. In the salivary glands of the vector, however, the coat is reacquired as the trypanosomes transform from epimastigote forms into the metacyclic stage which is infective to the mammalian host. This loss and acquisition of the surface coat can be correlated with the cyclical changes in net surface charge on the trypanosome which have been observed by other workers. The trypanosome populations of successive relapses in the blood are known to differ in their surface antigens (agglutinogens) and the loss of antigenic identity detected when any of these populations are put into culture indicates that these variable antigens are located in the surface coat. It is suggested that the coat in bloodstream trypanosomes constitutes a replaceable surface which, after being replaced, enables the trypanosome to escape the effects of host antibodies. The coat is therefore an adaptation to life in the bloodstream. Reacquisition of the surface coat by the metacyclic trypanosome after development in the vector may reflect reversion to a ‘basic’ antigenic type at this stage, preparatory to invading the blood of the mammalian host. The surface coat may be removed by the wide-spectrum proteolytic enzyme pronase, and this fact together with evidence from pH/mobility relationships and chemical analysis of the variable antigens suggest that the coat is basically proteinaceous. The coat may facilitate pinocytosis by binding proteins at sites within the pocket surrounding the base of the flagellum. In the non-pathogenic trypanosome T. lewisi a more diffuse filamentous coat is present in bloodstream forms and absent from culture forms. This trypanosome is said to carry a negative charge in both bloodstream and culture phases, so it seems likely that the nature of the coat in T. lewisi is different from that found in the pathogenic trypanosomes. In all these trypanosomes the flagellar membrane adheres to the surface membrane of the body throughout the life-cycle. Along the zone of adhesion lies a regular row of junctional complexes of the macula adherens type which, it is argued, serve in attachment. These attachments persist regardless of changes in the intervening cell surfaces.

Metabolic Co-Operation Between Biochemically Marked Mammalian Cells in Tissue Culture

Abstract: Cells of a genetic variant of the hamster fibroblast line BHK 21 which lack inosinic pyrophosphorylase activity ( IPP - cells) and therefore cannot normally incorporate [ 3 H]hypoxanthine were grown in mixed culture with cells of BHK 21 sublines which have inosinic pyrophosphorylase activity ( IPP + cells). If not in contact with IPP + cells, IPP - cells do not incorporate added [ 3 H]hypoxanthine into nucleic acid. IPP + cells always do incorporate [ 3 H]hypoxanthine and IPP - cells when in direct or indirect contact with IPP + cells also incorporate the isotope. Cell to cell contact appears to be essential for this gain of a metabolic function by IPP - cells. The possible molecular basis and general implications of the phenomenon are discussed.

Morphological variations in the dendritic spines of the neocortex.

Abstract: The structural variations which occur in the dendritic spines of pyramidal neurons of the somatic sensory cortex of the cat are described, particular attention being paid to spines attached to different parts of the dendritic tree. Spines may be recognized particularly by the absence of neurotubules and the common presence of a spine apparatus, and they can be considered as pedunculated or sessile , depending upon the presence or absence of a narrow pedicle. Within these 2 categories spines may be rounded, cup-like or prismatic, can be large or small, and may show various degrees of inversion of the surface receiving a synaptic contact. While spines of every size and shape may be attached to dendrites of all diameters, there is a definite tendency for the largest spines to occur on the smallest dendrites and vice versa. Furthermore, the smallest dendrites possess the spines with the longest pedicles. Every dendritic spine receives 1 axon terminal containing spherical synaptic vesicles and ending in an asymmetrical synaptic contact. In addition, 10-20% of the spines receive a second terminal, which in some cases may contain spherical vesicles and terminate asymmetrically but in others contains small, flattened or pleomorphic vesicles and ends in a symmetrical contact. The additional terminal may end on the pedicle of the spine or on the parent dendrite near the attachment of the pedicle. In the latter site, asymmetrical synapses are commonly associated with an additional spine apparatus.

Changes in The Cytochemical Properties Of Erythrocyte Nuclei Reactivated By Cell Fusion

Abstract: When the nucleus of a chick erythrocyte is introduced into the cytoplasm of a HeLa cell it resumes the synthesis of RNA and DNA. This reactivation of the red cell nucleus is associated with an increase in volume and with changes in nuclear composition. These changes have now been studied by quantitative cytochemical techniques. During the process of reactivation the dry mass of the erythrocyte nucleus shows a marked increase which takes place largely before the replication of the DNA begins. Within 24 h of cell fusion, some erythrocyte nuclei already contain an increased amount of DNA, and 48 h after fusion many of them contain twice the normal diploid amount, thus indicating that they have replicated their DNA completely. The physical properties of the nuclear deoxyribonucleoprotein complex also change. The ability of the nuclear chromatin to bind acridine orange increases 4- to 5-fold well before the synthesis of DNA begins; and changes in the melting profile of the deoxyribonucleoprotein suggest that its structure is loosened. This view is also supported by the observation that the reactivity of the erythrocyte nuclei to the Feulgen stain is altered during the early stages of reactivation.

The expression of genetic information: a study with hybrid animal cells.

Abstract: When the nucleus of a hen erythrocyte is introduced into the cytoplasm of a human or mouse cell in culture, it resumes the synthesis of RNA. The reactivated erythrocyte nucleus undergoes great enlargement, but it does not, for at least 2 or 3 days, develop nucleoli which can be discerned under the light microscope. During this period, the heterokaryon, although it may contain several active erythrocyte nuclei, does not synthesize any hen-specific surface antigens; and the hen-specific antigens introduced into the surface of the heterokaryon by the process of cell fusion are eliminated. But when, later, the erythrocyte nuclei do develop nucleoli, hen-specific antigens reappear on the surface of the heterokaryon and progressively accumulate. Before developing nucleoli, the erythrocyte nuclei synthesize little, if any, normal 28 S or 16 S RNA; but they do synthesize large amounts of the RNA which shows polydisperse sedimentation in conventional sucrose density gradients. Autoradiographic studies involving the use of a microbeam of ultraviolet light show, however, that this ‘polydisperse’ RNA is not transferred to the cytoplasm of the cell in detectable amounts so long as the erythrocyte nucleus lacks a definitive nucleolus. The inability of the erythrocyte nucleus at this stage to determine the synthesis of hen-specific surface antigens is thus attributable to the fact that it fails to transfer the RNA made on its chromosomes to the cytoplasm of the cell. When the erythrocyte nuclei develop nucleoli, however, the RNA which they make is transferred to the cytoplasm of the cell, and the synthesis of hen-specific surface antigens then begins. These experiments suggest that the nucleolus may play a decisive role in the transfer of information from nucleus to cytoplasm. The possible nature of this role is discussed.

Mitosis in Aspergillus nidulans.

Abstract: Mitosis in growing hyphae of haploid and diploid Aspergillus nidulans has been studied with the light microscope in Helly-fixed preparations. At the periphery of the nuleus a sharply defined granule is invariably found which has strong affinity for acid fuchsin. Mitosis begins with the duplication of this granule and the development inside the nucleus of a gradually lengthening fibre between the daughter granules. This fibre is also visibel in dividing nuclei of living hyphae examined by phase-contrast microscopy, when it appears as a grey bar traversing the non-nucleolar region. Division of the chromatin has been studied in preparations stained with aceto orcein, directly or after hydrolysis. It proceeds through four phases, beginning with the condensation of distinct chromosomes from the chromatin network of the resting nuclei. Later the chromosomes become arranged in two parallel rows of chromatinic masses in which individual chromosomes can no longer be distinguished. Division is completed by migration of the chromatin to the ends of each row, giving the appearance of a transverse break in the double bar. The chromatin accumulating at the ends of the double bar condenses to form the two daughter nuclei. The number of chromatinic elements appears to be the same in both haploid and diploid nuclei, although the individual elements are larger in the latter. Successive staining of the same dividing nucleus, first for the fibre (acid fuchsin) and then for the chromosomes (HCl-aceto orcein or HCl-Giemsa) has established that the fibre lies among the chromosomes, and that is elongation is closely related to the anaphase-telophase movements of the chromosomes. This suggests that the fibre is the equivalent of a mitotic spindle. That the fibre is connected with the chromosomes is further suggested by the observation that, in stained preparations, the fibre is considerably thicker in dividing diploid than in haploid nuclei. Electron microscopy of dividing nuclei has revealed that mitosis is carried out within the intact nuclear envelope. The mitotic spindle is composed of a bundle of fibrils which traverses the nucleus between two plaques of dense material associated with the envelope. In order to explain the configurations seen during the division of the chromatin it is proposed that the chromosomes become attached to the central spindle at various points along its length. With the elongation of the spindle the sister chromatids separate and pass, asynchronously, to opposite ends of the spindle along two preferred lines, thereby producing the characteristic double bar figure. The absence of a metaphase plate is thus accounted for by the scattered points of attachment of the chromosomes to the spindle and by their asynchronous division.

Phosphatases and differentiation of the Golgi apparatus.

Abstract: Cytochemical techniques for the electron microscopic localization of inosine diphosphatase, thiamine pyrophosphatase, and acid phosphatase have been applied to the developing root tip of Zea mays . Following formaldehyde fixation the Golgi apparatus of most of the cells showed reaction specificity for IDPase and TPPase. Following glutaraldehyde fixation marked localization of IDPase reactivity in the Golgi apparatus was limited to the root cap, the epidermis, and the phloem. A parallelism was apparent between the sequential morphological development of the apparatus for the secretion of a polysaccharide product, the fairly direct incorporation of tritiated glucose into the apparatus to become a component of this product and the development of the enzyme reactivity. Acid phosphatase, generally accepted as a lysosomal marker, was found in association with the Golgi apparatus in only a few cell types near the apex of the root. The localization was usually in a single cisterna at the face of the apparatus toward which the production of secretory vesicles builds up and associated regions of what may be smooth endoplasmic reticulum. Since the cell types involved were limited regions of the cap and epidermis and some initial cells, no functional correlates of the reactivity were apparent. Despite the presence of this lysosomal marker, no structures clearly identifiable as ‘lysosomes’ were found and the lack of reaction specificity in the vacuoles did not allow them to be so defined.

Muscle attachment related to cuticle architecture in Apterygota.

Abstract: In Apterygota muscles are attached to the cuticle by a series of discrete structures. The junction of the muscle and epidermal cells is demarcated by regular interdigitations of the two tissues, with desmosomes lining these processes. Within the epidermal cells, microtubules link up the desmosomes of the interdigitated region with dense material associated with cone-like depressions in the apical plasma membranes of the epidermal cells. Each of these ‘conical hemidesmosomes’ is situated opposite a pore canal. From within each cone, an electron-dense ‘muscle attachment fibre’ extends up the corresponding pore canal through the procuticle and is inserted on the epicuticle. There is no direct link between the microtubules and the muscle attachment fibres. The muscle attachment fibres are slightly elliptical in cross-section, and are twisted, this twist being in phase with the orientation of the chitin-protein microfibrils forming the lamellae of the procuticle. The attachment fibres are straight, and not helically arranged; patterns obtained in oblique sections of procuticle including these structures support the twisted ribbon model of pore canal shape. The cuticle, particularly in Thysanuran and Dipluran intersegmental membrane, displays the parabolic patterning typical of softer insect procuticle and procuticle deposition zones. The epicuticular insertion of the muscle attachment fibre is characterized by a pit in the cuticulin layer, the fibre passing through the middle of this pit. The microtubule-associated conical hemidesmosomes appear to be cytoskeletal in function. The muscle attachment fibres are rigid structures which are not digested by the moulting fluid enzymes. New muscle attachment fibres may only become attached to the epicuticle during its formation. The structures described in regions of muscle attachment in Apterygota are similar to those recorded for other arthropods.

Movement of cytoplasmic proteins into nuclei induced to enlarge and initiate DNA or RNA synthesis.

Abstract: In other studies it has been shown that somatic cell nuclei, which normally do not divide, are induced to enlarge and synthesize DNA when introduced into the cytoplasm of egg cells of Xenopus laevis. Introduction of such nuclei into the cytoplasm of large oocytes, however, causes nuclei to enlarge in a different way and to synthesize RNA but not DNA. In this study the proteins of eggs and oocytes were labelled with radioactive amino acids. Brain or blastula nuclei were then injected into cells containing labelled proteins under conditions in which protein synthesis was inhibited. Movement of cytoplasmic proteins was studied by observing the increase in acid-insoluble label over the injected nuclei by quantitative autoradiography. To prevent nuclear protein synthesis during the experiments, puromycin was injected with the nuclei. An estimation of the size of the labelled amino acid pool, a demonstration of the inhibitory effects of puromycin, and comparison of the amount of labelled material with and without puromycin all showed that protein synthesis in the nuclei played an insignificant role during the course of the experiments. A movement of acid-insoluble label from cytoplasm of egg cells into injected brain nuclei was noted even before they had begun to swell or synthesize DNA. In the initial period of nuclear enlargement there was a disappearance of the heterochromatic clumps characteristic of brain nuclei. This period coincided with a very rapid uptake of label to concentrations about twice that of the surrounding cytoplasm. A subsequent phase of nuclear swelling was characterized by a dilution of stainable nuclear material, loss of basophilia, and establishment of acidophilia of the nuclear contents. Cytoplasmic proteins continued to enter nuclei during this phase, but at a slower rate. Extraction of the soluble materials of labelled eggs with 0.01 M NaCl at pH 7.8 and a subsequent fractionation of the extract showed that there were many radioactive compounds present with molecular weights greater than 5000. The synthesis of DNA was initiated even before nuclear swelling could be detected, proceeding at least through the early stages of swelling. The induction of enlargement in blastula nuclei by oocyte cytoplasm containing labelled proteins was also accompanied by an uptake of label from the cytoplasm. In this case, although the uptake of unlabelled acidophilic material was much greater, the uptake of labelled proteins was much slower than that observed in the egg. The results are discussed in terms of chromosomal changes which occur during nuclear enlargement and during concomitant changes in nucleic acid metabolism.

Isopropyl N-Phenylcarbamate Affects Spindle Microtubule Orientation in Dividing Endosperm Cells of Haemanthus Katherinae Baker

Abstract: The mitotic inhibitor isopropyl N-phenylcarbamate (IPC) effects changes in the ultra-structure of the dividing endosperm cells of Haemanthus katherinae. In cells treated with 10 p.p.m. IPC for 0.5-2 h the microtubules lose their parallel alignment and become oriented in radial arrays. These radial arrays are interpreted as micropoles of the spindle apparatus and are thought to be the focal points for the chromosomes as they aggregate into micronuclei. Thus, IPC has a markedly different effect on the structure of the spindle apparatus than colchicine, which is observed at concentrations of 50 p.p.m. for 1 h to destroy the microtubules in Haemanthus. The structure of the microtubules appears unaffected by IPC since those from control and treated cells are similar in diameter (22 nm), in degree of staining, and in sensitivity to the fixation. In addition, the chromosomes and cytoplasmic components such as mitochondria, plastids, dictyosomes, elements of the endoplasmic reticulum, and ribosomes are not structurally modified by treatment with IPC.

Macromolecular Physiology of Plastids VII. The Effect of a Brief Illumination on Plastids of Dark-Grown Barley Leaves

Abstract: Barley seedlings developing at 23 °C in darkness have been studied at various ages for changes in fresh weight, height of shoot, protochlorophyll content and structure of the etioplasts in the primary leaf. Changes of the pigments in vivo , such as the spectral shift of the newly formed chlorophyll and the resynthesis of protochlorophyll, were studied spectrophotometrically prior to and following a 1-min illumination. Concomitantly, leaf tissue was fixed with glutaraldehyde-OsO 4 and later analysed in thin sections using the electron microscope. In darkness, protochlorophyll content in the primary leaf, size of the etioplasts, size of the crystalline prolamellar bodies and area of the primary lamellar layers reach their maximum values on the 7th day. Thereafter all the aforementioned parameters decrease, particularly the protochlorophyll content. The structural changes of the prolamellar-body material, rate of the spectral shift of the newly formed chlorophyll and rate of the resynthesis of protochlorophyll in darkness after photoconversion of the original protochlorophyll depend on seedling age. In both 5- and 7-day-old seedlings with high protochlorophyll content, the illumination causes a rapid transformation of the prolamellar bodies. Subsequently in 5-day-old seedlings, a rapid resynthesis of protochlorophyll takes place and the prolamellar bodies recrystallize before dispersal is completed. In the 7-day-old seedlings, resynthesis of protochlorophyll is slower and the prolamellar bodies are largely dispersed into primary lamellar layers before resynthesis of protochlorophyll and reformation of crystalline prolamellar bodies ensue. In 5-day-old seedlings, in which resynthesis of protochlorophyll and recrystallization of the prolamellar bodies are completed within less than 1 h in darkness following illumination, a second illumination effects a more rapid transformation and dispersal of the prolamellar bodies than is observed after the first illumination. In 9- and 11-day-old seedlings, in which the content of protochlorophyll is low, transformation of the prolamellar bodies occurs slowly in darkness following the initial illumination and is not completed within 60 min. No significant protochlorophyll resynthesis and no dispersal of the prolamellar bodies were observed during this time interval. Dispersal of the prolamellar bodies results in an increase in the area of the primary lamellar layers. During reformation of protochlorophyll, a decrease in area of the primary lamellar layers is correlated with an increase in volume and number of the prolamellar bodies. These quantitative relationships between the parameters for prolamellar bodies and primary lamellar layers indicate that, for dispersal and recrystallization of the prolamellar bodies, no significant amount of membrane synthesis is required. Different configurations of the prolamellar bodies can occur: protochlorophyll is associated with the crystalline configurations where the spacing of the tubules is either narrow or wide; chlorophyll is associated with the transformed prolamellar-body configuration. The narrow and wide crystalline configurations appear to be interconvertible, and re-organization does not necessarily involve the primary lamellar layers as an intermediate step. Wide spacing of the tubules coincides with the highest pigment content.

Linear synthesis of sucrase and phosphatases during the cell cycle of Schizosaccharomyces pombe.

Abstract: Department of Statistics, University of Edinburgh, ScotlandSUMMARYThe synthesi osf sucrase, acid phosphatas ande alkaline phosphatas haes been followe indsynchronous culture of ths e fission yeas Schizosaccharomycest pombe prepare bd y gradientsedimentation. These three enzymes follo a lineaw r pattern of synthesis through the cell cycle,with a doubling in rate at a 'critical point' about one-fift of thhe way throug thh e cycle.Sucrase can be rapidl y derepresse byd lowering the glucose concentratio inn the medium.This has bee n use tdo measure the sucrase ' potential' or capacity to synthesize sucras onederepression. The potential exist at alsl times in the cycle, and follow a stepwiss e pattern witha sharp ris aet the critical point.These results suggest that the functional genome doubles at the critical point. Since, however,the perio od f DNA synthesi iss nearly one-thir od f a cycle before this point, there b muse atnappreciable delay between chemical replicatio and functionanl replicatio of then genome. Inthis respec S.t pombe, a eukaryotic cell, differs markedly from bacteria.Other physiological events take place near the critical point, an ad tentative model is suggestedof what ma bye happening at the chromosomal level.Experiments with cycloheximide indicate that i thers a delaey between the synthesis an thedappearance of the active enzym ien the case of sucrase and alkaline phosphatase.

Observations on the Fine Structure of the Eyespot and Associated Organelles in the Dinoflagellate Glenodinium Foliaceum

Abstract: The eyespot of the marine dinoflagellate Glenodinium foliaceum is a flattened orange structure, more or less trapezoid in shape with an anterior hook-like projection. It is situated on the ventral side of the organism in the vicinity of the flagellar bases at the anterior end of the sulcus. In the electron microscope the eyespot is seen to contain two layers of osmiophilic granules 80-200 nm in diameter which usually show hexagonal close-packing. The eyespot is surrounded by a triple-membraned envelope and is not connected to any other organelle. Adjacent to the eyespot is a distinctive organelle termed the ‘lamellar body’. This consists of a stack of up to 50 flattened vesicles or disks, each 16 nm thick and about 750 nm wide, the whole being orientated in an antero-posterior direction. The lamellae are continuous, at the ends of the stack, with rough endoplasmic reticulum and are joined together by occasional bridges at their edges. The bases of the two flagella lie just ventral to the lamellar body and from them roots arise which pass by the eyespot and join the subthecal microtubular system. The eyespot of Glenodinium is unique both in structure and the presence of the associated lamellar body. It differs from eyespots which have been described from other algal groups and also from the more complex ocellus found in certain dinoflagellates belonging to the order Warnowiaceae. The method by which the eyespot functions is discussed and it is suggested that unidirectional stimuli could be perceived by shading of the lamellar body.

The energetics of mammalian cell growth.

Abstract: SUMMARY Data from batch growth curves of mouse LS cells cultivated at controlled dissolved oxygen partial pressures were used to calculate the weight of cells produced per mole of adenosine triphosphate generated (YArv). These values agree well with those reported for bacteria. A theoretical relationship was developed which allowed the biosynthetic and maintenance energy requirements to be estimated. The biosynthesis of LS cells required i-6 x io" 11 moles of ATP/cell. The maintenance energy, which is a function of growth rate, was 2-9 x io"" 11 moles ATP/new cell when the mean generation time was 1-15 days. The proportion of the total energy used for maintenance under these conditions was 65 %. This corresponds to a value of less than 10 % for bacterial maintenance when the organisms are grown at near their maximum rate. A comparison of biosynthetic energy requirements indicates that bacteria and moulds require about 4 times as much energy as animal cells to generate the same weight of cell material. Possible explanations of this difference are discussed.

The structure and some properties of the isolated mitotic apparatus.

Abstract: The morphology of the isolated sea-urchin mitotic apparatus (MA) was examined by light and electron microscopy. With the polarization microscope and the Nomarski differential interference microscope, the isolated MAs appeared to be similar to in vivo MAs. Electron microscopy of the isolated MAs revealed the presence of microtubules, ribosome-like particles and vesicles. A close association between the ribosome-like particles and the MA microtubules resulted in the appearance of chains of particles running along the length of the microtubules. Isolated MAs washed two to three times in isolation medium showed fine-structural changes in the electron microscope, which were reflected by lower retardation values obtained with the polarization microscope. The addition of magnesium and calcium or sucrose to the washing medium prevented these structural changes. Varying the pH of the isolation medium also resulted in changes in birefringence and ultrastructure of unwashed MAs. Isolated MAs stored in the original isolation medium gradually became less birefringent and lost their microtubules. At pH 6.1 and pH 6.2 a residual birefringence was retained, even after several weeks of storage. Electron microscopy of these MAs revealed the presence of linear aggregates of ribosome-like particles oriented parallel to the long axis of the spindle. On the other hand, at pH 6.3and pH 6.4, MAs lost their birefringence completely, and the ribosome-like particles became more randomly dispersed. 2M sucrose or 0.003 M Mg 2+ greatly retarded the loss of birefringence in stored MAs. Glutaraldehyde-fixed MAs stained intensely with azure B bromide, demonstrating the presence of RNA. Treatment with RNase resulted in a loss of this staining. RNase-treated MAs examined with the electron microscope, revealed changes in the ribosome-like particles. The results are discussed in the light of recent biochemical analyses of the isolated MA, structural similarities to in situ MAs and the interpretation of the birefringence of the MA.

Synthesis and Deposition of Oocyte Envelopes (Vitelline Membrane, Chorion) and the Uptake of Yolk in the Dragonfly (Odonata: Aeschnidae)

Abstract: Light and electron-microscope studies on dragonfly ovarioles reveal evidence that the precursor vitelline membrane and chorion secretions are synthesized within the follicle cells. It is suggested that the sequence of synthesis and deposition of the vitelline membrane occurs as follows. The vitelline membrane presecretion appears to be synthesized by the rough surfaced endoplasmic reticulum, giving rise to intracisternal granules. These appear to migrate in the cisternae to the region of the Golgi complex where the endoplasmic reticulum loses most of its ribosomes and the intracisternal granules move into the Golgi region where they appear within small vesicles. These seem to find their way into the Golgi cisternae where they may be incorporated with the secretions from the Golgi cisternae to produce the definitive previtelline secretion. The previtelline secretion bodies are eventually discharged into the space between the oocyte and follicle cells, forming rows of secretion bodies between the microvilli. These fuse into progressively larger bodies until a complete membrane is established. Follicle cells actively secreting precursor vitelline membrane substance show many disk-shaped, relatively clear vesicles in the cytoplasm. After the vitelline membrane is laid down, the follicle cells take on an entirely different function; namely, the synthesis and deposition of the chorion. The first visible chorion secretion appears in profile as elongate dense bodies within the Golgi cisternae which tend to coil, and in so doing, expand the cisternae. As this occurs, the enlarged cisterna, loaded with concentric coiled secretion material, separates from the remainder of the Golgi cisternae and becomes free in the cytoplasm as a prechorion secretion body. These migrate to, and collect below, the surface of the cell where they are eventually ejected between the surface folds and become incorporated into the developing chorion. Uptake of yolk in the dragonfly seems to be predominantly by micropinocytosis. The oocyte surface during active vitellogenesis bears many pits which contain an extracellular material closely applied to the outer surface of the plasma membrane. Thin, radially oriented bristles are continuous with the inner surface of the plasma membrane in this region. The pits continue to invaginate until they are cut off from the plasma membrane and come to lie in the oocyte cortex as coated vesicles. These appear to lose their coats gradually and fuse with one another to produce definitive yolk spheres.

Observations on the fine structure and development of the spindle at mitosis and meiosis in a marine centric diatom (Lithodesmium undulatum). II. The early meiotic stages in male gametogenesis.

Abstract: SUMMARY Various cytoplasmic phenomena, including spindle structure and development during prophase of the first meiotic division, are described and illustrated. The living culture is represented by a timed sequence of photographs continuing those previously published with respect to mitotic stages in the same filament. The meiotic preliminaries include the so-called swelling phase, by which the parental frustule is forced open, liberating the contained spermatocytes. This occurs during pachytene on evidence of chromosome structure which is illustrated. A spindle precursor is shown to be present before opening of the frustule; this resembles structurally the mitotic equivalent though the ground plan is oblong instead of square. Growth of the precursor continues until after opening of the frustule, when the spindle itself begins to be laid down. Two stages of developing spindles during the later prophases are illustrated by sections cut in three planes and by serial sections. Preliminary comparisons are made with metaphase I and with mitosis, both qualitatively and quantitatively, but a full discussion is deferred pending completion of the record for the later meiotic stages.

Observations on the Collagen and Proteinpolysaccharide Complex of Rabbit Corneal Stroma

Abstract: The form and interrelationship of the collagen fibrils and proteinpolysaccharide complex of rabbit corneal stroma were studied by electron microscopy. The intact tissue was examined as Araldite sections stained with alkaline lead citrate and uranyl acetate, and the mechanically disintegrated cornea after positive or negative staining with phosphotungstic acid or after treatment with 0.5% bismuth nitrate in 0.1 M nitric acid. The corneal collagen fibrils vary in cross-sectional area from 4.6 to 9.6 x 10 4 sq. A and do not exhibit a regular hexagonal distribution. Like tendon fibrils they consist of longitudinal filaments, but their appearance suggests that they lack some of the interfilament cross-links present in tendon. In sections of intact cornea and in negatively stained disintegrated cornea, filaments which are considered to be the protein cores of proteinpolysaccharide macromolecules are evident. They are about 40 A wide and 2000 A long. They appear to run an angular course, orthogonal to the collagen fibrils, and to be tangentially attached to several fibrils in the region of the a band. After treatment with bismuth nitrate disintegrated cornea contains coarsely beaded filaments. The filaments are about 2000 A long and the beads about 70 A in diameter. It is considered that these are again proteinpolysaccharide macromolecules and that each bead represents one or more polysaccharide chains in coiled configuration.

Preprophase Microtubule Bands in Some Abnormal Mitotic Cells of Wheat

Abstract: Caffeine treatment of growing wheat tissues was used to form binucleate or polyploid cells; preprophase microtubules in subsequent division cycles in these and some other abnormal cells were then examined. In root tips, binucleate cells or those with greatly enlarged nuclei usually contained one transverse preprophase band of microtubules; sometimes this was slightly asymmetrical or skew, and less commonly two bands were seen. In coleoptile vascular bundles, there were generally two or more bands in the greatly elongated cells, these sometimes appearing in different planes. During formation of the stomatal complexes, preprophase microtubules were almost invariably found where expected, preceding abnormal development both in untreated and also in caffeine-treated material, regardless of the number, disposition or size of nuclei. This occurred even when wall stumps, formed during a previous abortive division, indicated that that previous division was also asymmetrical. It is concluded that the position(s) of preprophase band(s) of microtubules is not particularly influenced by the nucleus or nuclei, being more susceptible to external morphogenetic influences which can persist for some considerable time. Particularly in the case of stomatal complexes, a cell wall seems necessary to seal off or otherwise fulfil the tendency towards asymmetrical division.

The classes of endosymbiont of paramecium aurelia

Abstract: The endosymbionts of Paramecium aurelia appear to consist of a number of different Gramnegative bacteria which have come to live within many strains of paramecia. It is not known whether in nature this relationship is mutually beneficial or not. The symbionts from one paramecium may kill other paramecia lacking that kind of symbiont. We identify the following classes of endosymbiotic organisms. First, kappa particles (found in P. aurelia , syngens 2 and 4) ordinarily contain highly characteristic refractile, or R, bodies, which are associated with the production of a toxin which kills sensitive paramecia. In certain mutants of kappa found in the laboratory both R bodies and ability to kill have been lost. Second, mu particles (in syngens 1, 2 and 8) produce the phenomenon of mate-killing. Third, lambda (syngens 4 and 8) and sigma particles (syngen 2) are very large, flagellated organisms which kill only paramecia of syngens 3, 5 and 9, and are enclosed in membrane-bound vacuoles. Fourth, gamma particles (syngen 8) are minute endosymbionts, surrounded by an additional membrane resembling endoplasmic reticulum. They have strong killing activity but no R bodies. Fifth, delta particles (syngens 1 and 6) possess a dense layer covering the outer membrane. At least one of the two known stocks is a killer. Sixth, nu particles are a heterogeneous group of particles (syngens 2 and 5) which do not kill or possess distinctive morphological characteristics. Seventh, alpha particles (syngen 2) are the only known nuclear symbionts of P. aurelia; they are found in the macronucleus. Alpha is also exceptional in being the only particle which is highly infectious, though certain of the other symbionts can also be taken up by paramecia lacking them, under special conditions.

Endocytosis of sugars in embryonic skeletal tissues in organ culture. IV. Lysosomal and other biochemical effects. General discussion.

Abstract: Cultivation of limb-bone rudiments in medium containing various non-metabolizable or poorly metabolizable sugars caused vacuolation of the perichondrial and articular cells and was accompanied by an increased synthesis and profuse secretion of lysosomal enzymes. Experiments with [14C] dextran indicated that the cytoplasmic vacuolation was due not to a higher rate of endocytosis, but rather to an abnormally long persistence of the pinocytotic vacuoles. Metachromatic material was lost from the cartilage only in the immediate vicinity of the vacuolated articular chondrocytes and the amount of hexosamine and hydroxyproline released into the medium was not much increased. However, the hexosamine and hydroxyproline of the sucrose treated rudiments was much more susceptible to extraction with neutral salt than those of paired controls. Sucrose taken up by the cells was liberated when the rudiments were returned to normal medium; this release, unlike the secretion of lysosomal enzymes, was unaffected by the presence of hydrocortisone. It is probable that the primary lysosornes, formed in the enlarged Golgi region, are the vehicles for the secretion of lysosomal enzymes. No appreciable amount of enzyrnic activity was released into the medium from rudiments grown in the presence of 0.08 M sucrose, before 36 h; at, or just before this time, however, a net increase in synthesis of enzyme was observed. The role of endocytosis in the resorption of skeletal matrix is discussed.

Synapses on the Axon Hillocks and Initial Segments of Pyramidal Cell Axons in the Cerebral Cortex

Abstract: The axon hillocks and initial segments of pyramidal cell axons can be clearly recognized in electron micrographs of the somatic sensory cortex. The initial segment is characterized by three features: bundles of neurotubules linked together by electron-dense bands, a layer of dense material attached to the inner surface of the plasma membrane, and small membrane-bound dense bodies. All of these elements and the few ribosomes usually present disappear at the commencement of the myelin sheath. The initial segment of the axon often contains a cluster of cisternae similar to the spine apparatus, and this part of the axon sometimes gives off small branches. Axon terminals end on both the axon hillock and the initial segment, and there is an increase in number on the latter as the distance from the hillock increases. All of these terminals are relatively large, contain a high proportion of small flattened or pleomorphic synaptic vesicles and terminate in symmetrical synaptic contacts. These morphological features suggest that the synapses may be inhibitory in function.

Synthesis of an enzyme determined by an erythrocyte nucleus in a hybrid cell.

Abstract: When a chick erythrocyte nucleus is introduced into the cytoplasm of a mutant mouse cell lacking the enzyme inosinic acid pyrophosphorylase, synthesis of the enzyme is induced. However, this synthesis does not begin until the erythrocyte nucleus develops a nucleolus. The kinetics of synthesis of the enzyme are essentially similar to those previously described for the synthesis of surface antigens specified by the chick nucleus. The results of both sets of experiments indicate that the nucleolus plays a critical role in the transfer of information from the genes to the cytoplasm of the cell.

Evidence For a Polarized Movement of the Lateral Loops of Newt Lampbrush Chromosomes During Oogenesis

Abstract: Actinomycin D inhibits RNA synthesis on the lateral loops of newt lampbrush chromsomes. Partial inhibition does not provoke marked morphological alteration of ordinary lateral loops, most of which recover to the full their capacity for RNA synthesis within 2 days of treatment. However, occasional ordinary loops do not recover completely within the first few days after treatment, and in such loops RNA-synthesizing capacity is restricted to a region adjoining the thinner insertion in the parent chromomere. A greater degree of inhibition of RNA synthesis is accompanied by loss of matrix from ordinary lateral loops, and in the extreme case the loop axes retract to their parent chromomeres and neighbouring chromomeres coalesce; for the ordinary loops, full recovery from this stripped condition is nevertheless possible. Some 20 µ per loop extends during the first day following exposure to actinomycin, and normal morphology and RNA-synthesizing capacity are regained within 2-4 days. The giant granular loop of Triturus cristatus cristatus chromosome XII responds to extreme actinomycin D poisoning in different fashion. Matrix does not at once slough off its loop axis, but the loop present at the time of treatment is progressively replaced by a new granular loop which develops between the parent chromomere and the original loop9s dense tip. These observations support the theory that the DNA-containing axes of all lateral loops of lampbrush chromosomes continually extend from their parent chromomeres, engage in RNA synthesis while extended, and carry the associated RNP matrix along as they move towards the return insertions in the parent chromomeres, where loop axis retraction occurs.

The Effect of Dissolved Oxygen Partial Pressure on the Growth and Carbohydrate Metabolism of Mouse LS Cells

Abstract: Batch cultures of mouse LS cells were grown in suspension at controlled dissolved oxygen partial pressures ( p O 2 . At low p O 2 (1.6 mmHg) the growth and respiration rates and the final cell population were all limited. At high p O 2 (320 mmHg), cell division was inhibited after an initial doubling of the cell number. At intermediate values of p O 2 the growth rate was constant but the final cell population varied. Within the p O 2 range of 40-100 mmHg, the final cell population was constant and maximal at 1.2x10 6 viable cells/ml. Except at 320 mmHg p O 2 about 90% of the glucose consumed served as an energy source and could be accounted for as lactate and CO 2 . In the culture at 320 mmHg, only 60% of the glucose consumed could be accounted for in this way. During growth the production of lactate and pyruvate was highest at low p O 2 . A sharp increase in lactate production was observed as logarithmic growth ceased in each culture, except at high p O 2 (160 mmHg). These observations indicate that p O 2 markedly influences cell growth and carbohydrate metabolism in these cells.

DNA synthesis and mitosis in fused cells. II. HeLa-chick erythrocyte heterokaryons.

Abstract: When chick erythrocyte nuclei are introduced into the cytoplasm of HeLa cells they resume the synthesis of DNA. The reactivated nuclei synthesize DNA in a highly co-ordinated manner and essentially in synchrony with the HeLa nuclei. Asynchronous DNA synthesis is rare. The erythrocyte nuclei in the HeLa cytoplasm respond to the signals which regulate DNA synthesis in the same way as the HeLa nuclei; and they do not, for at least 2 days after cell fusion, interfere in any way with the synthesis of DNA in the HeLa nuclei.

The uptake of tritiated uridine and phenylalanine by the ovaries of rats and monkeys.

Abstract: Rats and monkeys ( Macaca mulatta, M. irus ) were injected with tritiated uridine or phenylalanine and ovarian tissue was recovered at intervals from 15 min to 24 h later. Autoradiographs were prepared and studied by light microscopy; in addition some specimens which received uridine were examined using the electron microscope. The two isotopes are taken up by the ovaries of both species. The trace obtained with phenylalanine shows a very general distribution, whereas uridine seems to be more selectively incorporated by the various components of the ovary. The nuclei of primordial and growing oocytes are labelled with uridine, and so are the granulosa cells in follicles at all stages of development. The theca surrounding multilayered and vesicular follicles also shows a trace which appears heavier than that over the ovarian stroma. In the neonatal rat, the nuclei of oocytes at the pachytene and diplotene stages take up uridine. Autoradiographs prepared from ovaries fixed from 30 min to 4 h after injection and examined under the electron microscope show silver grains associated with the nucleoli and electron-dense chromosomal cores which are characteristic of these stages in meiotic prophase. Oocytes in primordial follicles, in immature and mature rats, are also labelled, showing that nuclear uptake continues as oocytes enter the ‘resting’ or dictyate stage. Since the chromosomes lose their morphological identity at this time, however, silver grains appear to be randomly distributed over the nuclear matrix. Primordial oocytes in the monkey also have a heavy nuclear label, seen under the light microscope to be associated with chromosomes and nucleoli. At the ultrastructural level, most of the extra-nucleolar silver grains are associated with condensed fibrillar material thought to represent part of the lateral component of chromosomes which are of the ‘lampbrush’ type. The synthetic activity of germinal and somatic elements in the ovary is discussed, and possible differences in the structure and metabolic activity of oocyte chromosomes are considered in relation to intra- and inter-specific variations in radiosensitivity.

Microtubules and filaments in the filopodia of the secondary mesenchyme cells of Arbacia punctulata and Echinarachnius parma.

Abstract: In an attempt to understand the mechanism of contraction of the filopodia of the secondary mesenchyme cells and thus secondary invagination of the archenteron, the fine structure of these processes was examined. Whereas microtubules are commonly encountered in the cell body and at the base of the filopodia, very few (one or two) are present near the tip of the filopodia. Instead the slender processes are filled with 50-A filaments. Colchicine and hydrostatic pressure were applied to the embryos to elucidate the action of these different fibrous elements. Both agents cause cessation of archenteron movement and the disassembly of the microtubules. Hydrostatic pressure causes the disappearance of the filaments as well. Because of the small numbers of microtubules in the slender filopodia and the fact that in no other system is there any evidence for contraction of these elements, it was concluded that they do not function in the contraction process, but are probably involved in the formation of these cell extensions: hence the effect of colchicine on archenteron movement. The 50-A filaments, on the other hand, are likely candidates for the contraction process.

The Ultrastructure and Ontogeny of Pollen in Helleborus Foetidus L : III. The Formation of the Pollen Grain Wall

Abstract: The first recognizable elements of the pollen grain wall of Helleborus foetidus are initiated in the cellulosic primexine which is formed immediately outside the microspore cytoplasm while the pollen grains are still in the tetrad configuration and enveloped in a thick layer of callose. Elements of the primexine give rise to the precursors of the rod-like bacula of the mature exine. The bacula increase in electron density due to the rapid deposition of sporopollenin, and begin to expand laterally at the outer side to form the tectum. There follows a lateral expansion on the inner side to form the foot-layer. Further deposition of sporopollenin is continued and all elements of the pollen grain wall expand outwards and laterally as the pollen grain enlarges. The enveloping callose disappears and the pollen grains are free in the thecal cavity. A secondary exine is deposited below the primary exine, particularly around the furrows. Initially this process involves a number of thin electron-transparent lines or lamellae about 4 nm thick that appear to arise from the cytoplasm and provide a locus around which sporopollenin is deposited. As the deposition proceeds, the lamellae thicken and finally merge with each other to form the secondary exine. No sign of the lamellae can be seen in the mature pollen grain wall. Towards the end of secondary exine formation the deposition of sporopollenin does not appear to be centred on thin lamellae, but appears as small granules which gradually coalesce. The secondary exine remains discontinuous in the region of the furrow, but becomes consolidated in the inter-furrow regions. As the pollen grain matures the sporopollenin, which is electron-dense when initially deposited, becomes progressively less so. The final stage in development is the deposition of the cellulosic intine, which forms inside the secondary exine and is associated with increased dictyosome activity and randomly oriented microtubules.

Alteration of Nuclear Distribution in B-Mutant Strains of Schizophyllum Commune

Abstract: Sexual morphogenesis in Schizophyllum commune , a higher basidiomycete, is controlled by two incompatibility factors, A and B . A key event, the migration of nuclei from each mate throughout the mycelium of the other, is controlled by the B factor and occurs in A = B ≠ matings. The distribution of nuclei in the resulting heterokaryon is irregular, and anucleate, uninucleate, binucleate and multinucleate cells are found. A similar distribution of nuclei is found in homokaryons carrying a mutation in the B factor. Because of their developmental history, strains that carry a mutated B factor offer a relatively simple system for the study of the events associated with nuclear migration. Growth of mutant- B germlings occurs in three stages: (I) most cells are binucleate; (II) most cells are uninucleate; (III) cells contain varied numbers of nuclei. The ratio of nuclei; cells remains constant during the transition from stage II to stage III. Changes in nuclear distribution result from movement of the nuclei from cell to cell, and the movement is associated with the disruption of the dolipore septum. The mutant- B system appears to offer an opportunity for the biochemical resolution of the events related to nuclear migration.