Protoplasmic streaming in the giant unicellular green algaAcetabularia mediterranea

Summary Protoplasmic Streaming inAcetabularia mediteranea has been studied by microcinematography in 1. germinating zygotes, 2. germlings before the differentiation of rhizoids and apices, 3. young cells with rhizoids and apices, 4. vegetative cells-several centimeters in length, 5. cells with a maximum sized cap, containing secondary nuclei, and 6. cells after cyst formation. Intracellular transport is found to occur at a network-system of thin filaments and at a different system of headed streaming bands. At the network of filaments chloroplasts are found to move at a velocity of 1–2 m/sec. Headed streaming bands move along the filaments and may lead without interruption from the rhizoid to the apex of the cell andvice versa. The front zone of the streaming bands is occupied by a leading cytoplasmic head-structure. Small vesicles, polyphosphate granula and secondary nuclei are the predominant moving structures in headed streaming bands. The velocity of these particles is found to be 3–11 m/sec. The filament system is found during all developmental stages. Headed streaming bands are undetectable in germinating zygotes and develop from small cytoplasmic droplets in germlings to broad heavily loaded bands in the huge vegetative cell.Transport of secondary nuclei by headed streaming bands is not observed during mitotic divisions and after cyst formation, though moving bands are still present for several weeks after cyst formation.     

Electron spin resonance study of the isolated lipid components from Blastocladiella emersonii zoospores

The physical properties of the plasma membrane of the aquatic phycomycete Blastocladiella emersonii were investigated, in particular the effects of cations on membrane structure. Intact zoospores and lipid extracts were labelled with the spin-labels 5-nitroxystearate (5-NS), 12-nitroxystearate (12-NS), and 2,2,6,6-tetramethylpiperidine-1-oxyl (Tempo). Electron spin resonance spectroscopy indicated a total of three breaks in plots of the hyperfine splitting parameter, 2T_|, order parameter, S, and the partition coefficient, f, vs. temperature. The first and third break points (T_L and T_H) were found to be independent of the external K⁺, Ca²⁺, or Mg²⁺ concentrations. They were similar to the break points found in aqueous dispersions of lipid extracts and correlate well with the temperature limits for zoospore viability. In contrast, the middle break point (T_M) was markedly influenced by the external Ca²⁺ concentration. Ca²⁺ increased T_M from 12°C (no Ca²⁺ added) to 22°C (10 mM Ca²⁺), i.e., growth temperature. K⁺ reversed this Ca²⁺ effect, downshifting T_M from 22°C to 10°C. A comparison of the physico-chemical effects of these ions on the membrane, as revealed by the cation-induced shift in T_M, is closely correlated with the temperature dependence and physiological effects of cations on zoospore differentiation. This suggests that cations may modify the physical state of the plasma membrane and be involved in regulating the initial changes during zoospore encystment.     

Isozyme differentiation of aldolase and pyruvate kinase in fetal,regenerating, preneoplastic,and malignant rat hepatocytes during culture

Summary Aldolase and pyruvate kinase isozymes were investigated in cultured hepatocytes from fetal, regenerating, and 2-acetyl-aminofluorene-fed rat liver as well as in some epithelial liver cell lines. Our results show that: (a) cell proliferation and prolonged expression of specific isozymes were found only in cultured hepatocytes from 17-day old fetuses; (b) the fetal type of pyruvate kinase expressed in regenerating and carcinogen-treated liver was temporarily lost only in cultured hepatocytes from regenerating liver; (c) the adult type of aldolase and pyruvate kinase was absent in one epithelial cell line derived from a carcinogen-treated liver and in the hepatoma tissue cell (HTC) line but was found in the Faza clone of the Reuber H₃₅ cell line during the 50 first passages in vitro; and (d) the isozyme pattern of pyruvate kinase was always more strongly shifted than that of aldolase. The observations suggest that: (a) hepatocytes from carcinogen-treated liver exhibit the same lack of ability to proliferate in primary culture as normal adult hepatocytes; (b) adult hepatocytes can produce fetal isozymes without prior cell division; (c) pyruvate kinase is a stronger marker of dedifferentiation (retrodifferentiation) than aldolase; and (d) regulatory processes of isozyme expression are different during ontogenesis, regeneration, and hepatocarcinogenesis. This work was supported by the “Institut National de la Santé et de la Recherche Médicale” and the “Fondation pour la Recherche Medicale Fran?aise”     

Membrane-fusions and cytoplasmic bridges in the cells of the developing cerebellum

Summary In the developing cerebellum of the neonate rats membranefusions and cytoplasmic bridges between cells were observed. These membrane-fusions were characterized by the presence of loops of membrane and cytoplasmic bridges between the two limits of the membrane-fusions. They were found between Purkinje cells, Purkinje cells and the migratory cells, mitotically potent cells of the external granular layer, and differentiating granule cells of the internal granular layer. The membrane-fusions were found to be a transient developmental phenomenon. Issues pertaining to the universality of membrane-fusions, their significance in the induction for cell differentiation, and the problem of fixation artifacts are discussed.This research was supported by N.I.H. Research Grants No. NS-08817 and CA-14650. Assistance of Mrs. Kunda Das in various aspects of electron microscopy is gratefully acknowledged     

Maintenance and induction of morphological differentiation in dissociated mammary epithelium on floating collagen membranes

Summary Dissociated normal mammary epithelial cells from prelactating mice were plated on different substrates in various medium-serum-hormone combinations to find conditions that would permit maintenance of morphological differentiation. Cells cultured on floating collagen membranes in medium containing insulin, hydrocortisone and prolactin maintain differentiation through 1 month in culture. The surface cells form a continuous epithelial pavement. Some epithelial cells below the surface layer rearrange themselves to form alveolus-like structures. Cells at both sites display surface polarization; microvilli and tight junctions are present at their medium-facing or luminal surface and a basal lamina separates the epithelial components from the gel and stromal cells. Occasinal myoepithelial cells, characterized by myofilaments and plasmalemmal vesicles, are identified at the basal surface of the secretory epithelium. In contrast, cells cultured on plastic, glass or collagen gels attached to Petri dishes form a confluent epithelial sheet showing surface polarization, but lose secretory and myoepithelial specializations. If these dedifferentiated cells are subsequently maintained on floating collagen membranes, they redifferentiate. There is little DNA synthesis in cells on collagen gels, in contrast to Petri-dish controls. Protein synthesis in cells on floating collagen membranes increases over T₀ values and remains constant through 7 days in culture whereas it decreases on attached gels; however, if the gels are freed to float, protein synthesis increases sharply and parallels that seen on floating membranes. The work was supported by USPHS Grants CA-05388 and CA-05045 from the National Cancer Institute, DHEW.     

Fetal mouse lung in circumfusion system cultures

Summary Fetal mouse lungs were cultivated, using the dual-rotary circumfusion system for tissue culture, and their histotypic development was surveyed for 75 days by phase-contrast and electron microscopy. Alveoli, terminal bronchioles and alveolar macrophages were photographed periodically with still and time-lapse phase-contrast microscopy. Their histotypic appearance was confirmed by electron micrographs of the 1- and 2 1/2-month-old specimens. These revealed typical alveoli surrounded by a basal lamina and composed of types I and II pneumocytes containing various lamellar-body forms within the type II cells, the alveolar lumen, and the alveolar macrophages. There was a shift from almost all type II cells in the 1-month-old alveoli to the presence of frequent type I cells as constituents of the alveoli in the 2 1/2-month-old cultures. The terminal bronchioles were tubules consisting of ciliated cells with Clara cells interspersed between them. The ciliated cells contained as many as 30 cilia or basal bodies per section and numerous microvilli. They were attached to each other and to the Clara cells by junctional complexes and accessory desmosomes which were generally in the apical ends of the cells. The Clara cells typically had glycogen granules interspersed between lamellae of the endoplasmic reticulum, contained numerous well dispersed mitochondria, occasional lysosome-like granules and crystalloid bodies which appeared to be tubular. Some Clara cells presented a moderately dense secretory granule in the center of the whorl of the endoplasmic reticulum. This work supported by Grant HL19684 from the National Heart and Lung Institute, National Institutes of Health. Pregnant Strong A mice were kindly supplied by Dr. Henry Browning of the Department of Anatomy.     

Growth and morphology of neuronal cell lines cultured in perfusion

Summary To optimize culture conditions and gain a more reliable culturing system for studies of metabolic properties of neuronal cells, a simplified perfusion chamber was developed. It consists of two parts: a perfusion block and a standard plastic culture dish. To confirm the suitability of this chamber for continuous culturing of anchorage-dependent cells, the growth and morphology of the four neuronal cell lines glioma C6 and glioma 138MG, neuroblastoma C1300, clones N1E115 and N18 were followed for 4 d using both traditional and perfusion techniques. A marked increase in growth and a decrease in the degree of morphological differentiation were obtained with the latter technique compared to the former. This work was supported by grants from the National Swedish Board for Technical Development (Grant 81-5009), the Swedish Work Environmental Foundation (Grant 76-53), and Ollie and Elof Ericssons Foundation for Scientific Research.     

Differences in blastema-associated proteins according to position along body axis in regenerating planarians

To test the hypothesis that neoblasts in different positions in regenerating pieces of planarians may be differentially responsive to diffusible stimuli through differentially expressed membrane receptors, we compared membrane surface proteins in blastemas induced at various positions along an anterior-posterior axis in Dugesia gonocephala (Dugès). The proteins were biotinylated and identified by molecular weight in SDS-PAGE (sodium-dodecylsulfate polyacrilamide gel electrophoresis). This SDS-PAGE pattern was then compared with that of N-glycosylated proteins incorporating ³H-mannose. One 20 kDa glycoprotein present in all blastemas at 3 d was absent from more anterior blastemas at 6 d irrespective of whether those blastemas were at the cephalic or the caudal end of the regenerating piece. The expression of this protein appears to be determined by the position of the blastema along the body axis rather than by its prospective fate.     

Isolation of a Repeated DNA Probe Showing Polymorphism among Meloidogyne incognita Populations

Several Meloidogyne incognita geographic populations were characterized by analysis of the restriction fragment length polymorphisms (RFLP) obtained after digestion of their total DNA and hybridization with a [³²P]-labeled probe. The probe consisted of a 1.7-kb-repeated DNA sequence, isolated from a M. incognita genomic library, that hybridized to multiple BamH I fragments in the genome of each isolate. The patterns showed sufficient polymorphism to enable the accurate differentiation of all the populations tested.