REJUVENATION OF MELOSIRA GRANULATA (BACILLARIOPHYCEAE) RESTING CELLS FROM THE ANOXIC SEDIMENTS OF DOUGLAS LAKE,MICHIGAN. I. LIGHT MIGROSCOPY AND 14C UPTAKE 1

Resting cells of Melosira granulate (Ehr.) Ralfs were collected from the anoxic sediments of Douglas Lake, Michigan. Sediment containing M. granulata was inoculated into distilled water and incubated in a growth chamber for one week during which observations were made on the cytological differentiation process. Cells classified as “condensed,” i.e. containing a dark brown cytoplasmic mass were identified as resting cells. The differentiation process consisted of a series of gradual cytological changes that included elongation of the cytoplasmic mass and recognition of definable organelles to the point where the cells were non-distinguishable from water column vegetative cells. Differentiating cells accumulated large polyphosphate and lipid granules. However, these granules disappeared just prior to cell division. The complete differentiation or rejuvenation sequence occurred in some cells in less than 24 h. However, not all dormant cells rejuvenated at the same time and it was observed that the lag period for rejuvenation increased with resting cell age (depth of burial in sediments). In the ¹⁴C uptake studies, label was initially observed in condensed state cells. The label gradually progressed to the more differentiated forms. Total carbon uptake during the rejuvenation process was initially lower in the rejuvenating cells, but roughly equal to water column populations after 8 h, indicating a period of high metabolic activity in the rejuvenating cells between 1 and 8 h.     

Diatom resting cell rejuvenation and formation: time course, species records and distribution

Physiological resting cells (as opposed to resting spores orcysts) have been identified for the following freshwater diatomspecies: Actinocydus normanii f. subsalsa, Asterionella formosa,Diatoma tenue var. elongatum, Fragilaria capucina, F.construens,F.construens var. venter, F.crotonenss, F.intermedia var. fallax,F.pinnata, Melosira granulata, M.islandica, M.italica subsp.subarctica, Stephanodiccus alpinus, S.binderanus, S.medius,S.niagarae, Tabellaria fenestrata and T.flocculosa. Restingcell populations were obtained from surficial sediments of threebays of the Laurentian Great Lakes. Surficial sediment resuspensions(down to 3 cm) were carried out in filtered bay water underlighted conditions which resulted in rejuvenation of these speciesto growing vegetative populations. All resting cells were characterizedby a dense cytoplasmic mass positioned in the center of thecell. Density of this mass varied between species. The abilityof individual species to rejuvenate is affected by temperature,and probably nutrients and/or other environmental factors. Restingcell formation was studied in a unialgal culture of M.granulataisolated from a Douglas Lake resuspension. Resting cells appearas a function of culture age; however, their formation can begreatly accelerated by reduced temperatures and darkness. Ourobservations suggest that the ability to form resting cellsand entrainment of such cells through turbulent mixing is animportant factor in determining phytoplankton community structureand succession in the Great Lakes.     

Viability of phytoplankton resting stages in the sediments of a coastal Swedish fjord

Viable diatom and dinoflagellate resting stages were recovered from sediments in Koljö Fjord on the west coast of Sweden. To determine the maximum survival time of buried resting stages, samples from sediment depths down to 50?cm were incubated at temperatures of 3, 10 and 18?°C. Sediment cores were dated by ²¹⁰Pb and the age of samples containing viable resting stages was determined using the constant rate of supply model. Dilution cultures of surface sediments allowed semiquantitative estimates of the potential seed bank. Dinoflagellate cysts from species such as Diplopsalis sp., Gymnodinium nolleri, Oblea rotunda and Protoceratium reticulatum were viable down to 15?cm depth, or 37 years old. Spores and resting cells of the diatoms Chaetoceros spp., Detonula confervacea and Skeletonema costatum were viable to over 40?cm depth, and may have been buried for many decades. The seed bank of living resting stages in surficial sediments was found to be rich (c. 57000 diatom resting stages g^?1 wet weight and c. 200 dinoflagellate cysts g^?1 wet weight), and the percentage of viable resting stages was higher for spore- and cyst-forming species. The oxygen-deficient sediments in Koljö Fjord appear to be a natural conservator of cell viability, a condition not easily simulated in laboratory studies. These results are ecologically important since spores and cysts are a repository of genetic material able to repopulate waters if resuspended and exposed to suitable light, temperature and nutrients.     

Expression of Epstein-Barr viral capsid, complement fixing, and nuclear antigens in stationary and exponential phase cultures

Three continuous lymphoblastoid cell lines, 2 productive of nucleocapsids and 1 nonproductive line, were studied for their content of Epstein-Barr viral (EBV) antigens during transition from stationary to logarithmic phase growth. As a preliminary step, viable cells were separated from degenerating ones in discontinuous gradients of serum albumin. Viral capsid antigens were found in both living and dead cells of the 2 producer lines; however, complement fixing (CF) antigens and nuclear antigen were detected only in viable cell subpopulations. The content of antigen detectable in extracts of viable cells by complement fixation remained constant in replicating and resting cultures; further, all viable cells of the 3 lines demonstrated intranuclear antigen by anticomplement immunofluorescence in all stages of growth. In contrast, the proportion of cells with viral capsid antigen in the producer lines increased 7- to 24-fold following entry of resting populations into the phase of exponential growth.The results suggest that expression of viral capsid antigens is discontinuous and is initiated in response to events in log phase, possibly DNA synthesis or mitosis. Expression of the complement fixing and nuclear antigens in continuous in viable cells. These findings emphasize the intimate relationship of the CF and nuclear antigen to the transformed state and suggest that study of this antigen complex will shed light on the mechanisms of lymphocyte transformation by EBV.     

褶纹冠蚌精子发生的研究

光镜和透射电镜研究结果表明：褶纹冠蚌精子发生是非同步的,精子发生经历了一系列重要的形态和结构变化,主要包括：核逐步延长、染色质浓缩、线粒体逐渐发达与融合、胞质消除以及鞭毛的形成。精原细胞胞质中含有许多致密的轴纤丝,它们后来形成鞭毛轴丝。精母细胞质中含有线粒体、中心粒、内质网和电子透明的囊泡。精细胞分化为４个时期。成熟精子属原始类型,由头部、中段和尾部三部分组成。多核结构和细胞间桥自始至终存在于精子     

Distribution of myoepithelial cells and basement membrane proteins in the resting, pregnant, lactating, and involuting rat mammary gland

Using antisera to specific proteins, the localization of the rat mammary parenchymal cells (both epithelial and myoepithelial), the basement membrane, and connective tissue components has been studied during the four physiological stages of the adult rat mammary gland, viz. resting, pregnant, lactating, and involuting glands. Antisera to myosin and prekeratin were used to localize myoepithelial cells, antisera to rat milk fat globule membrane for epithelial cells, antisera to laminin and type IV collagen to delineate the basement membrane and antisera to type I collagen and fibronectin as markers for connective tissue. In the resting, virgin mammary gland, myoepithelial cells appear to form a continuous layer around the epithelial cells and are in turn surrounded by a continuous basement membrane. Antiserum to fibronectin does not delineate the basement membrane in the resting gland. The ductal system is surrounded by connective tissue. Only the basal or myoepithelial cells in the terminal end buds of neonatal animals demonstrate cytoplasmic staining for basement membrane proteins, indicating active synthesis of these proteins during this period. In the secretory alveoli of the lactating rat, the myoepithelial cells no longer appear to form a continuous layer beneath the epithelial cells and in many areas the epithelial cells appear to be in contact with the basement membrane. The basement membrane in the lactating gland is still continuous around the ducts and alveoli. In the lactating gland, fibronectin appears to be located in the basement membrane region in addition to being a component of the stroma. During involution, the alveoli collapse, and appear to be in a state of dissolution. The basement membrane is thicker and is occasionally incomplete, as also are the basket-like myoepithelial structures. Basement membrane components can also be demonstrated throughout the collapsed alveoli.     

Rehabilitation of cancer through oncogene inactivation

The inactivation of the MYC oncogene alone can reverse tumorigenesis. Upon MYC inactivation, tumors stereotypically reverse, undergoing proliferative arrest, cellular differentiation and/or apoptosis. The precise consequences of MYC inactivation appear to depend upon both genetic and epigenetic parameters. In some types of cancer following MYC inactivation, tumor cells become well differentiated and biologically and histologically normal, inducing sustained tumor regression. However, in some cases, these normal-appearing cells are actually dormant tumor cells and upon MYC reactivation they rapidly recover their tumorigenic properties. Future therapies to treat cancer will need to address the possibility that tumor cells can camouflage a normal phenotype following treatment, resting in a dormant, latently cancerous state.     

Structure of human chromosomes visualized at the electron microscopic level

A newly developed technique allows cytological (light microscope level) chromosome preparations to be examined at the electron microscopic level. Ultrathin (50 nm) sections of highly condensed Hela cell metaphase chromosomes show the characteristic mitotic chromosome morphology. In addition a fibrous network (presumably chromosome fibers) can be seen within them. Fibers appear to be gathered at foci along each chromatid. Treatment of chromosomes with trypsin in a trypsin/G-banding procedure reduces the amount of staining material at the electron microscopic level and results in more prominent foci. Thicker (100 nm) sections of less condensed chromosomes prepared from human lymphocytes display a banding pattern similar to G-banding, even without pretreatment with proteases.     

Cytological characterization of isolated sperm cells of perennial ryegrass (Lolium perenne L.)

Summary The cytoplasmic content and the distribution of intramembrane particles (IMPs) of the plasma membrane of isolated sperm cells of perennial ryegrass (Lolium perenne L.) have been characterized using flow cytometry, transmission electron microscopy, confocal scanning laser microscopy and freeze-fracture studies. The isolated haploid sperm cells contain a variety of cell organelles with the exception of microtubules. Proplastids and plastids with starch were observed, although only rarely. Vacuoles containing remnants of organelles and stacked lamellae of endoplasmic reticulum with cytoplasmic inclusions were observed frequently, indicating that autophagy takes place. The number of mitochondria varies from 11 to 26 with an average of 17. Generally, the nucleus has a lobed shape and displays various interphasic stages of chromatin condensation. The analysis of the number of mitochondria and the nuclear state did not show evidence of sperm cell dimorphism. The cytological variability observed, could be explained by differences in developmental stages already present in vivo at the moment of isolation. No correlation between the number of mitochondria and the nuclear cross-sectioned area and/or the condensation state of the chromatin could be found. The density of intramembrane particles of the plasma membrane on the exoplasmic fracture face is more than twice that on the protoplasmic fracture face. That is the opposite of what was found for sporophytic cells of perennial ryegrass. These results are discussed in relation to the potential use of these cells for biotechnology and developmental studies.     

Reversible calcium-regulated stopcocks in legume sieve tubes

Sieve tubes of legumes (Fabaceae) contain characteristic P-protein crystalloids with controversial function. We studied their behavior by conventional light, electron, and confocal laser scanning microscopy. In situ, crystalloids are able to undergo rapid (<1 sec) and reversible conversions from the condensed resting state into a dispersed state, in which they occlude the sieve tubes. Crystalloid dispersal is triggered by plasma membrane leakage induced by mechanical injury or permeabilizing substances. Similarly, abrupt turgor changes imposed by osmotic shock cause crystalloid dispersal. Because chelators generally prevent the response, divalent cations appear to be the decisive factor in crystalloid expansion. Cycling between dispersal and condensation can be induced in opened cells by repetitive exchange of bathing media containing either Ca(2)+ or chelators. Sr(2)+ and Ba(2)+, but not Mg(2)+, are equally active. In conclusion, the fabacean P-protein crystalloids represent a novel class of mechanically active proteinaceous structures, which provide an efficient mechanism with which to control sieve tube conductivity.     

Membrane organization determines barrier properties of endothelial cells and short-chain sphingolipid-facilitated doxorubicin influx

The endothelial lining and its outer lipid membrane are the first major barriers drug molecules encounter upon intravenous administration. Our previous work identified lipid analogs that counteract plasma membrane barrier function for a series of amphiphilic drugs. For example, short-chain sphingolipids (SCS), like N-octanoyl-glucosylceramide, effectively elevated doxorubicin accumulation in tumor cells, both in vitro and in vivo, and in endothelial cells, whereas other (normal) cells remained unaffected. We hypothesize here that local membrane lipid composition and the degree of lipid ordering define SCS efficacy in individual cells. To this end, we study the differential effect of SCS on bovine aortic endothelial cells (BAEC) in its confluent versus proliferative state, as a model system. While their (plasma membrane) lipidome stays remarkably unaltered when BAECs reach confluency, their lipids segregate to form apical and basolateral domains. Using probe NR12S, we reveal that lipids in the apical membrane are more condensed/liquid-ordered. SCS preferentially attenuate the barrier posed by these condensed membranes and facilitate doxorubicin influx in these particular membrane regions. We confirm these findings in MDCK cells and artificial membranes. In conclusion, SCS-facilitated drug traversal acts on condensed membrane domains, elicited by confluency in resting endothelium.     

Comparative study of the elemental composition of vegetative and dormant microbial cells

X-ray microanalysis showed that vegetative cells, viable resting forms, and nonviable forms (micromummies) of the bacteria Bacillus cereus and Micrococcus luteus and the yeast Saccharomyces cerevisiae differ in the contents of bioelements S, P, Ca, and K and the Ca/K and P/S ratios. Viable resting forms (cystlike refractory cells and bacillar endospores) had more calcium and less phosphorus and potassium than vegetative cells, the difference being higher for bacilli than for micrococci and yeasts. The distinctive feature of all viable resting microbial forms was their low P/S ratios and high Ca/K ratios. The differences revealed in the cellular content and ratios of bioelements probably reflect changes in ionic homeostasis accompanying the transition of vegetative microbial cells to the dormant state. Relevant potassium parameters indicate that the membranes of viable resting forms retain their barrier function. At the same time, the nonviable forms, even morphologically intact, of B. cereus and S. cerevisiae exhibited an anomalously low content of potassium, while those of M. luteus had an anomalously high content of this element. This suggests that the cellular membranes of micromummies lose their barrier function, which results in a free diffusion of potassium ions across the membranes. The possibility of using the elemental composition parameters for quick analysis of the physiological state of microorganisms in natural environments is discussed.     

Peltate trichomes in the dormant shoot apex of Metrodorea nigra,a Rutaceae species with rhythmic growth

• In Metrodorea nigra, a Rutaceae species with rhythmic growth, the shoot apex in the dormant stage is enclosed by modified stipules. The young organs are fully covered with peltate secretory trichomes, and these structures remain immersed in a hyaline exudate within a hood-shaped structure. Our study focused on the morpho-functional characterization of the peltate trichomes and cytological events associated with secretion.
• Shoot apices were collected during both dormant and active stages and processed for anatomical, cytochemical and ultrastructural studies.
• Trichomes initiate secretion early on, remain active throughout leaf development, but collapse as the leaves expand; at which time secretory cavities start differentiation in the mesophyll and secretion increases as the leaf reaches full expansion. The subcellular apparatus of the trichome head cells is consistent with hydrophilic and lipophilic secretion. Secretion involves two vesicle types: the smaller vesicles are PATAg-positive (periodic acid/thiocarbohydrazide/silver proteinate) for carbohydrates and the larger ones are PATAg-negative. In the first phase of secretory activity, the vesicles containing polysaccharides discharge their contents through exocytosis with the secretion accumulating beneath the cuticle, which detaches from the cell wall. Later, a massive discharge of lipophilic substances (lipids and terpenes/phenols) results in their accumulation between the wall and cuticle. Release of the secretions occurs throughout the cuticular microchannels.
• Continued protection of the leaves throughout shoot development is ensured by replacement of the collapsed secretory trichomes by oil-secreting cavities. Our findings provide new perspectives for understanding secretion regulation in shoot apices of woody species with rhythmic growth.

     

Measurement of cytoplasmic fluorescence depolarization of single cells in a flow system.

We have built a flow system in which we can analyze and sort individual viable cells on the basis of their cytoplasmic microviscosity. The average cytoplasmic microviscosity (or cytoplasmic structuredness) of a cell can be quantitated by exciting fluorescein molecules in the cytoplasm of the cell with a polarized light source and measuring the extent of depolarization of the fluorescent signal. Changes in the state of a cell (e.g., the reception of a signal inducing the cell to differentiate) often appear to be associated with changes in cytoplasmic microviscosity, these changes being detectable within hours of the inducing signal. An example of one such change is presented.     

Ultrastructural and lanthanum tracer examination of rapidly resorbing rat alveolar bone suggests that osteoclasts internalize dying bone cells

Glutaraldehyde-formaldehyde fixed undecalcified alveolar bone from 7-day-old rats was prepared for light and electron microscopy. Colloidal lanthanum was used as an ultrastructural tracer, and both random and semi-serial sections were examined. Lanthanum penetrated the infoldings of the ruffled border and some nearby vacuoles and vesicles. The majority of vacuoles and vesicles were lanthanum-free. Some osteoclast profiles contained a large vacuole with a cell enclosed in its interior. The enclosed cell exhibited an irregular nucleus containing condensed peripheral chromatin, intact cytoplasmic organelles, conspicuous rough endoplasmic reticulum and large blebs on the cell surface. These features are characteristic of osteoblasts or bone-lining cells or immature osteocytes which may be undergoing apoptosis or necrosis. The observation of remnants of cellular structures within internalized osteoclast vacuoles, together with the above results, suggests that osteoclasts engulf and probably degrade dying osteoblasts/bone-lining cells or immature osteocytes. Received: 29 April 1997 / Accepted: 20 February 1998     

Comparative Study of the Elemental Composition of Vegetative and Resting Microbial Cells

X-ray microanalysis showed that vegetative cells, viable resting forms, and nonviable forms (micromummies) of the bacteria Bacillus cereus and Micrococcus luteus and the yeast Saccharomyces cerevisiae differ in the content of elements S, P, Ca, and K and Ca/K and P/S ratios. Viable resting forms (cystlike refractive cells and bacillar endospores) had more calcium and less phosphorus and potassium than vegetative cells, the difference being higher for bacilli than for micrococci and yeasts. The distinctive feature of all viable resting microbial forms was their low P/S ratios and high Ca/K ratios. The differences revealed in the cellular content and ratios of elements probably reflect changes in ionic homeostasis accompanying the transition of vegetative microbial cells to the dormant state. Relevant potassium parameters indicate that the membranes of viable resting forms retain their barrier function. At the same time, the nonviable micromummies, even those morphologically intact, of B. cereus and S. cerevisiae exhibited an anomalously low content of potassium, while those of M. luteus had an anomalously high content of this element. This suggests that the cellular membranes of micromummies lose their barrier function, which results in a free diffusion of potassium ions across the membranes. The possibility of using the elemental composition parameters for the quick analysis of the physiological state of microorganisms in natural environments is discussed.     

The encystment of a freshwater dinoflagellate: A light and electron-microscopical study

The process of encystment, or resting spore formation, in a freshwater dinoflagellate (Woloszynskia tylota nov. comb.) has been studied with both light and electron microscopy. The main features of the process are as follows: (i) the replacement of the theca by a thin, amorphous outer wall, which gradually thickens by the deposition of material on its inner face; (ii) the appearance of a layer of closely-packed lipid droplets at the cytoplasmic margin of the mature cyst, resembling a granular ‘inner wall’ in the light microscope; (iii) the reduction in size or disappearance of cytoplasmic structures such as chloroplasts, Golgi bodies and pusule; and (iv) the enlargement of a central ‘accumulation body’ and cytoplasmic vacuoles containing crystals. Comparisons are made with light-microscope studies of encystment of other dinoflagellates, with ultrastructural studies of non-motile division stages, with zooxanthellae and with fossil dinoflagellate cysts or hystrichospheres.     

FINE STRUCTURAL ALTERATIONS OF INTERPHASE NUCLEI OF LYMPHOCYTES STIMULATED TO GROWTH ACTIVITY IN VITRO

This report describes fine structural changes of interphase nuclei of human peripheral blood lymphocytes stimulated to growth by short-term culture with phytohemagglutinin. Chromatin is found highly labile, its changes accompanying the sequential increases of RNA and DNA synthesis which are known to occur in lymphocyte cultures. In "resting" lymphocytes, abundant condensed chromatin appears as a network of large and small aggregates. Early in the response to phytohemagglutinin, small aggregates disappear during increase of diffuse chromatin regions. Small aggregates soon reappear, probably resulting from disaggregation of large masses of condensed chromatin. Loosened and highly dispersed forms then appear prior to the formation of prophase chromosomes. The loosened state is found by radioautography to be most active in DNA synthesis. Small nucleoli of resting lymphocytes have concentric agranular, fibrillar, and granular zones with small amounts of intranucleolar chromatin. Enlarging interphase nucleoli change chiefly (1) by increase in amount of intranucleolar chromatin and alteration of its state of aggregation and (2) by increase in granular components in close association with fibrillar components.     

THE FINE STRUCTURE OF GREEN BACTERIA

The fine structure of several strains of green bacteria belonging to the genus Chlorobium has been studied in thin sections with the electron microscope. In addition to having general cytological features typical of Gram-negative bacteria, the cells of these organisms always contain membranous mesosomal elements, connected with the cytoplasmic membrane, and an elaborate system of isolated cortical vesicles, some 300 to 400 A wide and 1000 to 1500 A long. The latter structures, chlorobium vesicles, have been isolated in a partly purified state by differential centrifugation of cell-free extracts. They are associated with a centrifugal fraction that has a very high specific chlorophyll content. In all probability, therefore, the chlorobium vesicles are the site of the photosynthetic apparatus of green bacteria.     

CHROMATOGRAPHIC ANALYSES OF ACID-SOLUBLE POLYPHOSPHATES IN CHLORELLA CELLS

Using uniformly ³²P-labeled Chlorella cells as material, compositionof acid-soluble inorganic polyphosphates was studied by paperchromatography and ion-exchange chromatography. 2.By the paper chromatographic analysis it was found that theacid-soluble polyphosphates consisted of highly condensed polyphosphates.Ring-forming tri- and tetrametaphosphates, pyrophosphate andtripolyphosphate were not detected in the acid-soluble fractionof the algal cells. 3.By an ion-exchange chromatography with the use of increasingconcentrations of KCl-solution as eluant, it was found thatthe acid-soluble polyphosphate was a mixture of polyphosphateswith a variety of condensation number (n-values). Polyphosphatesof the n-values between 3 and 15 were only 20% of the totalacid-soluble polyphosphate. The majority of the other polyphospateshad greater n-values which was eluted with 0.5–1.0 M KCl. (Received March 2, 1964; )