Regeneration of proximal and distal body part in hydra exposed to ultraviolet light (2535 angstroms)

Pelmatohydra oligactis was amputated in the central part of the gastral region and exposed to radiation of ultraviolet rays (2535 angstroms, 12 erg mm(-2)s(-2) for 7, 15 and 20 minutes. The regeneration of the proximal and distal part of the animal which was fixed 8, 24, 48, 72 and 96 hours after the cutting and radiation has been studied cito-histologically. The regeneration of the wounds caused by cutting and those caused by radiation have been compared. It has been found out that the wounds caused by radiation heal much harder and that the radiation-destroyed hypostome needs a longer period to regenerate than the cutting-removed hypostome. It is assumed that radiation-destroyed parts have an inhibitory effect upon environment. But, cito-histological changes concord to a great degree in both cases. The foot regeneration in the animals cut and exposed almost entirely concords the regeneration in the control animals which were cut but not exposed. Namely, both of them, as a rule, remain permanently without a foot. In the paper we have tried to explain these results and brought out the conclusion that hydras do not regenerate the foot because in the bud region there are many zimogen and interstitial cells which are not characteristic of a foot and that is why hydra has a directed growth exclusively toward the distal part, never the opposite. The growth is localized to the hypostome and the bud region. Radiation does not inhibit the process of the budding that has already begun. It is assumed that undamaged cell material travels from the gastroderm toward the bud and serves its formation.     

Automated quantification of budding Saccharomyces cerevisiae using a novel image cytometry method

The measurements of concentration, viability, and budding percentages of Saccharomyces cerevisiae are performed on a routine basis in the brewing and biofuel industries. Generation of these parameters is of great importance in a manufacturing setting, where they can aid in the estimation of product quality, quantity, and fermentation time of the manufacturing process. Specifically, budding percentages can be used to estimate the reproduction rate of yeast populations, which directly correlates with metabolism of polysaccharides and bioethanol production, and can be monitored to maximize production of bioethanol during fermentation. The traditional method involves manual counting using a hemacytometer, but this is time-consuming and prone to human error. In this study, we developed a novel automated method for the quantification of yeast budding percentages using Cellometer image cytometry. The automated method utilizes a dual-fluorescent nucleic acid dye to specifically stain live cells for imaging analysis of unique morphological characteristics of budding yeast. In addition, cell cycle analysis is performed as an alternative method for budding analysis. We were able to show comparable yeast budding percentages between manual and automated counting, as well as cell cycle analysis. The automated image cytometry method is used to analyze and characterize corn mash samples directly from fermenters during standard fermentation. Since concentration, viability, and budding percentages can be obtained simultaneously, the automated method can be integrated into the fermentation quality assurance protocol, which may improve the quality and efficiency of beer and bioethanol production processes.     

Endosymbiotic bacteria associated with the intracellular green algae ofHydra viridis

Gram-negative bacteria 4.5–5.5 μm in length and 1.2 μm in diameter are found in gastrodermal cells of three stains of freshwater green hydras,Hydra viridis (Ohio and Carolina from North America, Jubilee strain from England). They are motile via single polar flagella. They were detected in live animals, Jensen stained material, and electron micrographic sections. Bacteria lose motility quickly upon release from hydra cells. Green hydras harbor strain-specific numbers of chlorellae in these cells. Other tissue types lack algae. The chlorella-hydra symbiosis can be disassociated and the partners grown separately; transfer of photosynthate from algae to hydra occurs. Here we report the presence of endocellular bacterial vesicles specifically associated with cells that contain the symbiotic chlorellae. No cells that contained algae and lacked bacteria were seen. Vesicles, especially conspicuous in sexually mature green hydras, are probably produced upon extrusion from the cell. They contain either algae and bacteria or bacteria alone and are often expelled to the surrounding medium via the coelenteron. Bacteria are absent in nerve cells, interstitial cells, nematocysts, mucous cells, sperm, and probably in most of the other cell types that lack algae. They are present in at least one cell type that lacked algae: the columnar ovarian cells. Bacteria were lost in “bleached” hydras, those induced to lose their algae by high intensity light in a solution of DCMU, a standard inhibitor of photosynthesis. They were absent in a fourth strain of green hydra (Connecticut Valley,H. viridis) and inH. fusca andH. littoralis, two freshwater nonsymbiotic hydras. All of the hydra lacking bacteria contain conspicuous lipid droplets in their cells. The presence of large numbers of bacteria has implications for interpretations of metabolic exchange between host and algal symbionts and for extrapolation of metabolic data from strain to strain ofH. viridis.     

The relationship between size,budding rate,and growth efficiency in three species of hydra

Three species of the fresh water carnivore hydra, H. littoralis, H. pseudoligactis, and C. viridissima present a graduation in size with the first species the largest and albino Chlorohydra the smallest. When presented with a daily overabundance of food (artemia), considerable variation in food intake and gross efficiency of growth (proportion of food energy consumed that is turned into new protoplasm or buds) existed among the species. The degree of association between size of species and food intake was highly significant. However, budding efficiency among the species was found to be independent of food intake (when the effects of species size were eliminated) and of species size (when the effects of food intake were removed). However, species with high (low) efficiencies have significantly higher (lower) reproductive rates. A lowering of the temperature from 25° to 15° C. increased the size of the species, increased food intake, but decreased reproductive rate. In all species except H. pseudoligactis a corresponding increase in the production of bud energy with no change in efficiency also occurred. On the other hand, lowering of the temperature for H. pseudoligactis significantly lowered reproductive efficiency but had no effect on the total calorific output of buds. This species, in constrast to the others, appears to have a compensatory ability to adjust its efficiency to maintain a high calorific output when temperature increases. It was also found that albino Chlorohydra have budding efficiencies of around 35 percent which are not influenced by changes in food intake or light. Normal green hydras, however, have efficiencies which range from 40 to 62 percent above their albino counterparts when fed once a day and once every two days in light respectively. It it concluded first, that the symbiotic algae in the gastrodermals cells of green hydra contribute quantitatively in the order of the above amounts to the growth process in this species, and second, that green hydras have the ablity to increase their growth efficiency when food intake is reduced thus reducing the drop in calorific but output that normally occurs in the albino (control) form.     

Involvement of cytoplasmic membranes in the non-lytic infection of K-562 cells by Semliki Forest virus

An analysis of the human leukemia cell line, K-562, infected with Semliki Forest virus, has been made with transmission electron microscopy. In contrast to the usual surface budding of the enveloped virus on the plasma membrane of vertebrate cells leading to cytolysis within 20 h, K-562 cells do not show surface budding, and the cells remain intact for periods of several months. Several unusual features of the infection include: 1) the rough endoplasmic reticulum arranges early into continuous perinuclear chains; 2) during the time of virus replication and release, the nucleocapsids aggregate on the cytoplasmic side of internal vesicles in the region of the cell where the Golgi complex is normally located; and 3) during this same time period, the vesicles are seen to contain enveloped virions and rod-like formations, a result suggesting that budding has occurred into these vesicles. Viruses are presumably released from the cell as these vesicles fuse with the plasma membrane. By 12 days post-infection and thereafter, the intact cells show electron-dense aggregates of chromatin, large vacuoles and lipid inclusions throughout the cytoplasm, and only a few virion-containing vesicles.     

Blocked coated pits in AtT20 cells result from endocytosis of budding retrovirions

AtT20 cells support the replication of two endogenous retroviruses, a murine leukemia virus and a mouse mammary tumor virus. On glass or plastic substrates, AtT20 cells grow in clumps. In this situation, retroviruses budding from the plasma membrane of one cell can, on rare occasions, be invested by coated pits in the plasma membranes of contiguous cells. These pits can invaginate to depths of 2,000-4,000 A within the cytoplasm drawing with them the viral buds which remain connected to their parental cells by tubular stalks, some of which are only 225 +/- 15 A in diameter. These stalks run down the straight necks of the pits from the buds to the parental cell surfaces. Several lines of evidence indicate that these unique structures are blocked such that neither endocytosis nor budding can go to completion, and that they persist for several hours. The properties of these blocked coated pits are relevant to models of both endocytosis and viral budding. First, they indicate that the invagination of a coated pit is not absolutely dependent on its pinching off to form a coated vesicle, but that uncoating appears to be dependent upon the generation of a free vesicle. Secondly, they suggest that the final stages in the maturation of a retroviral core into a mature nucleoid are dependent on the detachment of the bud from its parental cell and that the driving force of budding is the association of viral transmembrane proteins with viral core proteins. An explanation is offered to account for the formation of these structures despite the phenomenon of viral interference.     

Interstitial stem cells in Hydra: multipotency and decision-making

Interstitial stem cells in Hydra constitute a population of multipotent cells, which continuously give rise to differentiated products during the growth and budding of Hydra polyps. They also give rise to germ cells in animals undergoing sexual differentiation. Cloning experiments have shown that interstitial stem cells are multipotent. In vivo tracing of stem cell lineages has revealed that stem cells divide symmetrically to yield two stem cells or asymmetrically to yield one stem cell daughter and one daughter cell which initiates nerve or nematocyte differentiation. Following commitment, some nerve cell precursors migrate from the body column into the head or foot region, thus giving rise to the high density of nerve cells observed in these regions. Stem cell proliferation is regulated by changes in the self-renewal probability and is controlled by stem cell density. Nerve cell commitment is controlled by several peptides including the Head Activator. Factors affecting nematocyte commitment are not known, but wnt and notch signaling are both required for differentiation of committed precursors.     

Budding and strobilation inAurelia (Scyphozoa,Cnidaria): Functional requirement and spatial patterns of nucleic acid synthesis

Summary Strobilation and polypoid budding occur at different locations in the scyphistoma (polyp). Initiation and completion of both forms of budding are inhibited by hydroxyurea (HU), which blocks 95% of DNA synthesis within 12 h. Gradients of thymidine incorporation into both cell layers of the body column precede and accompany strobilation, and an epidermal gradient precedes polypoid budding. In both, the highest labelling index is in the zone in which initiation will occur. Polypoid buds show high variation in labelling index, which is therefore not significantly different from body column labelling. Initiation and some elongation of polyp buds occurs in a small percentage of animals in HU, indicating that cell recruitment is important for these processes. Strobilation appears to be more highly dependent on localized nucleic acid synthesis than polypoid budding.     

光周期对中国水螅种群增长、无性出芽生殖及抗氧化酶活力的影响

单细胞真核绿藻在中国水螅(Hydra sinensis)内胚层皮肌细胞中共生是有较高科研价值的特殊生物学现象。水螅宿主细胞为共生藻提供CO2、氮源及矿物质,而共生藻通过光合作用可能为宿主提供碳水化合物等有机物营养,因此水螅与共生藻间代谢流是以共生藻光合作用为中心,但基于代谢流二者间的互作机制目前尚未阐明。水螅通过营养积累进行出芽生殖,从母体脱落的芽体数量间接反映水螅营养积累的相对量。而光暴露时长能影响共生绿藻光合作用,如果共生藻的确能向水螅细胞转移光合作用产物,那光暴露时长应该能间接影响水螅的营养积累、从而进一步影响水螅无性出芽生殖。为证实该假说,本研究应用种群累积培养法,观察了光周期对中国水螅种群增长、无性出芽生殖及抗氧化酶(SOD和CAT)活力的影响。结果显示,光周期对中国水螅种群增长具有明显的影响。培养15 d后,所有实验组水螅的种群密度均为正增长,其中8L∶16D(在一个24h周期内光暴露8 h、黑暗16 h)实验组种群密度最大、而0L∶24D(持续黑暗)实验组种群密度最小。另外,随着光暴露时长的增加,中国水螅SOD及CAT活力整体均呈下降趋势。结果表明,从光周期对中国水螅无性出芽生殖及两种抗氧化酶活力的影响来看,中国水螅对光周期的生理学响应较为敏感,这个现象可能源于共生绿藻能通过向宿主细胞转移光合作用产物的方式为水螅提供营养补充。     

OBSERVATIONS ON SUPERNUMERARY HEAD FORMATION INDUCED BY LITHIUM CHLORIDE TREATMENT IN THE REGENERATING HYDRA, PELMATOHYDRA ROBUSTA

Lithium chloride treatment of hydras cut just proximal to the tentacle circle and just distal to the budding region induces a supernumerary head at the proximal cut surface. Such a supernumerary head does not appear in the normal course of regeneration. The bipolar hydra thus formed persists for several weeks and later separates to form two normal individuals. The supernumerary head is not formed at the proximal cut surface when the hydra is transected just distal to the budding zone and the distal portion is allowed to regenerate in the Li-containing medium. LiCl has a slight inhibitory effect on the regeneration of hypostomes or tentacles when the animal is cut at the base of the hypostome.     

Synchronous cell growth occurs upon synchronizing the two regulatory steps of the Saccharomyces cerevisiae cell cycle

There are two known asynchronous steps in the budding yeast Saccharomyces cerevisiae cell cycle, where an asynchronous step is one which is completed in different lengths of time by different cells in an isogenic population. It is shown here that elimination of the asynchrony due to cell size by preincubation of cells with the mating pheromone alpha-factor, and decreasing the asynchrony in the cdc28 'start' step by lowering the pH, yields highly synchronous cell growth measured as the time period between the emergence of buds. In one experiment, cell budding for 92% of cells occurred within a 12-min period for at least two generations. Under identical conditions, cell number increase is not as synchronous as bud emergence indicating that there is a third asynchronous step, which is concluded to be at cell separation. These results are consistent with there being two--and only two--asynchronous steps in the cell cycle, measured from bud emergence to bud emergence. Surprisingly, these two steps are also the two major regulatory steps of the cell cycle. It is concluded that asynchrony may be a general feature of cell cycle regulatory steps. The asynchrony in the completion of the cdc28 'start' step which occurs in the first cell cycle after alpha-factor washout is shown here to be almost or entirely eliminated for the second passage through this step after alpha-factor washout. The 'true' time between the onset of budding and the point where 50% of cells have budded (called t50BE) is 17 and less than or equal to 2 min for the first and second budding, respectively, after alpha-factor washout. The cell cycle models requiring a transition probability, or asynchrony, at 'start' for every cell cycle are therefore incorrect.     

Binding of the wheat germ lectin to Cryptococcus neoformans suggests an association of chitinlike structures with yeast budding and capsular glucuronoxylomannan

The capsule of Cryptococcus neoformans is a complex structure whose assembly requires intermolecular interactions to connect its components into an organized structure. In this study, we demonstrated that the wheat germ agglutinin (WGA), which binds to sialic acids and beta-1,4-N-acetylglucosamine (GlcNAc) oligomers, can also bind to cryptococcal capsular structures. Confocal microscopy demonstrated that these structures form round or hooklike projections linking the capsule to the cell wall, as well as capsule-associated structures during yeast budding. Chemical analysis of capsular extracts by gas chromatography coupled to mass spectrometry and high-pH anion-exchange chromatography suggested that the molecules recognized by WGA were firmly associated with the cell wall. Enzymatic treatment, competition assays, and staining with chemically modified WGA revealed that GlcNAc oligomers, but not sialic acids, were the molecules recognized by the lectin. Accordingly, treatment of C. neoformans cells with chitinase released glucuronoxylomannan (GXM) from the cell surface and reduced the capsule size. Chitinase-treated acapsular cells bound soluble GXM in a modified pattern. These results indicate an association of chitin-derived structures with GXM and budding in C. neoformans, which may represent a new mechanism by which the capsular polysaccharide interacts with the cell wall and is rearranged during replication.     

A role for the Pkc1p/Mpk1p kinase cascade in the morphogenesis checkpoint

In many cells the timing of entry into mitosis is controlled by the balance between the activity of inhibitory Wee1-related kinases (Swe1p in budding yeast) and the opposing effect of Cdc25-related phosphatases (Mih1p in budding yeast) that act on the cyclin-dependent kinase Cdc2 (Cdc28p in budding yeast). Wee1 and Cdc25 are key elements in the G2 arrest mediated by diverse checkpoint controls. In budding yeast, a 'morphogenesis checkpoint' that involves Swe1p and Mih1p delays mitotic activation of Cdc28p. Many environmental stresses (such as shifts in temperature or osmolarity) provoke transient depolarization of the actin cytoskeleton, during which bud construction is delayed while cells adapt to environmental conditions. During this delay, the morphogenesis checkpoint halts the cell cycle in G2 phase until actin can repolarize and complete bud construction, thus preventing the generation of binucleate cells. A similar G2 delay can be triggered by mutations or drugs that specifically impair actin organization, indicating that it is probably actin disorganization itself, rather than specific environmental stresses, that triggers the delay. The G2 delay involves stabilization of Swe1p in response to various actin perturbations, although this alone is insufficient to produce a long G2 delay.     

中国绿水螅分子系统发生地位的初步探讨

目的:初步探讨中国绿水螅(Hydra sinensis)分子系统发生地位以及水螅属内部各类群系统发生关系。方法:采用酚-氯仿法提取中国绿水螅总DNA,扩增线粒体COI和16S r RNA基因片段并进行DNA序列测定,再利用Clustal及MEGA等生物信息学分析软件进行系统发生分析。结果:在本研究重建的所有系统发生树中,中国绿水螅始终与绿水螅Hydra viridissima的不同种群一起构成绿水螅单系群。同时,棕色水螅群的单系性被基于COI基因的NJ树以及基于16S r RNA基因的NJ树和ML树支持,唯独基于COI基因的ML树不支持棕色水螅群的单系发生。在基于COI基因的ML树中纤弱水螅族在系统树的基部独立为一支系,而绿水螅群和其他棕色水螅群水螅一起组成另一支系,提示纤弱水螅族水螅的系统发生地位值得进一步探讨。值得注意的是,根据本文的结果,棕色水螅群内3族的划分仍然有一定疑问。基于COI基因的NJ树和ML树支持普通水螅族、寡水螅族和纤弱水螅族各自族内的单系发生,但16S r RNA基因的NJ树和ML树中仅普通水螅族水螅聚为单系群,而寡水螅族和纤弱水螅族水螅各自并非单系发生。结论:把水螅属划分为绿水螅群及棕色水螅群有一定的合理性,但棕色水螅群内寡水螅族、普通水螅族和纤弱水螅族3族的划分还有待商榷。     

Up-regulation of the Cdc42 GTPase limits the replicative life span of budding yeast

Cdc42, a conserved Rho GTPase, plays a central role in polarity establishment in yeast and animals. Cell polarity is critical for asymmetric cell division, and asymmetric cell division underlies replicative aging of budding yeast. Yet how Cdc42 and other polarity factors impact life span is largely unknown. Here we show by live-cell imaging that the active Cdc42 level is sporadically elevated in wild type during repeated cell divisions but rarely in the long-lived bud8 deletion cells. We find a novel Bud8 localization with cytokinesis remnants, which also recruit Rga1, a Cdc42 GTPase activating protein. Genetic analyses and live-cell imaging suggest that Rga1 and Bud8 oppositely impact life span likely by modulating active Cdc42 levels. An rga1 mutant, which has a shorter life span, dies at the unbudded state with a defect in polarity establishment. Remarkably, Cdc42 accumulates in old cells, and its mild overexpression accelerates aging with frequent symmetric cell divisions, despite no harmful effects on young cells. Our findings implicate that the interplay among these positive and negative polarity factors limits the life span of budding yeast.     

Apoptosis-like yeast cell death in response to DNA damage and replication defects

In budding (Saccharomyces cerevisiae) and fission (Schizosaccharomyces pombe) yeast and other unicellular organisms, DNA damage and other stimuli can induce cell death resembling apoptosis in metazoans, including the activation of a recently discovered caspase-like molecule in budding yeast. Induction of apoptotic-like cell death in yeasts requires homologues of cell cycle checkpoint proteins that are often required for apoptosis in metazoan cells. Here, we summarize these findings and our unpublished results which show that an important component of metazoan apoptosis recently detected in budding yeast-reactive oxygen species (ROS)-can also be detected in fission yeast undergoing an apoptotic-like cell death. ROS were detected in fission and budding yeast cells bearing conditional mutations in genes encoding DNA replication initiation proteins and in fission yeast cells with mutations that deregulate cyclin-dependent kinases (CDKs). These mutations may cause DNA damage by permitting entry of cells into S phase with a reduced number of replication forks and/or passage through mitosis with incompletely replicated chromosomes. This may be relevant to the frequent requirement for elevated CDK activity in mammalian apoptosis, and to the recent discovery that the initiation protein Cdc6 is destroyed during apoptosis in mammals and in budding yeast cells exposed to lethal levels of DNA damage. Our data indicate that connections between apoptosis-like cell death and DNA replication or CDK activity are complex. Some apoptosis-like pathways require checkpoint proteins, others are inhibited by them, and others are independent of them. This complexity resembles that of apoptotic pathways in mammalian cells, which are frequently deregulated in cancer. The greater genetic tractability of yeasts should help to delineate these complex pathways and their relationships to cancer and to the effects of apoptosis-inducing drugs that inhibit DNA replication.     

ULTRAVIOLET MICROSCOPY OF BUDDING SACCHAROMYCES.

McClary, Dan O. (Southern Illinois University, Carbondale), Wilbert D. Bowers, Jr., and Glendon R. Miller. Ultraviolet microscopy of budding Saccharomyces. J. Bacteriol. 83:276-283. 1962.-Synchronous cell division was obtained in Saccharomyces by transferring starved cells into nutrient medium. Ultraviolet microscopy and Giemsa-stained preparations of these cells showed nuclear division to occur in the mother cell early in the budding process. The divided nucleus passed into the neck between the mother cell and the bud, and either fused together again or the two parts became so closely associated that one continuous dumbbell-shaped body was seen which seemed to divide by constriction. This effect was probably due to the retention of the intact nuclear membrane until nuclear division was otherwise complete.The nuclear apparatus lies outside the vacuole. The extent to which these bodies function together cannot be determined by the techniques employed in this work.Parallel experiments on meristematic cells of onion root tips show the reliability of these cytological methods.     

Growth regulation of the interstitial cell population in hydra. II. A new mechanism for the homeostatic recovery of reduced interstitial cell populations

The interstitial cells of hydra comprise a stem cell population, producing at least two classes of terminally differentiated cell types, nerve cells and nematocytes. Exposure to hydroxyurea (HU) results in selective depletion of interstitial cells from the tissue. The surviving cells subsequently recovered to normal levels, and the mechanisms involved in this repopulation were examined. Hydra were treated for varying times with HU such that interstitial cell numbers were reduced to 7 or 35% of normal. Subsequent growth of the epithelial and interstitial cell populations in these animals was monitored. The results indicate that the growth rates of these two cell types were only slightly different from untreated controls during the 4 weeks after HU exposure, implying that repopulation should not have occurred. However, recovery of the interstitial cell population was observed. Further analysis revealed that the interstitial cells in HU animals, unlike normal hydra, were not uniformly distributed in the body column, and were especially reduced in the budding region. In normal animals a constant fraction of the interstitial and epithelial cells are lost into buds. However, as a consequence of this nonuniform distribution a smaller fraction of the interstitial cells are displaced into HU buds, thereby retaining a higher proportion in the adult tissue. Calculations indicate that this mechanism of increased retention is of sufficient magnitude to account for 40-60% of the observed recovery after HU treatment.     

Stammzellen in Hydra. Evolutionäres Vermächtnis

The basis for Hydra's enormous regeneration capacity and potential immortality is a life cycle in which proliferation occurs mostly asexual by budding. That requires that each polyp contains a large number of cells which continuously undergo self‐renewing mitotic divisions and also have the option to follow differentiation pathways. Now, emerging molecular tools for the first time shed light on the molecular processes controlling these pathways. Studies of stem cells in Hydra, therefore, promise critical insights of general relevance into stem cell biology and the evolutionary origin of these cells.     

Filament ring formation in the dimorphic yeast Candida albicans

Stationary phase cultures of Candida albicans inoculated into fresh medium at 37 degrees C synchronously from buds at pH 4.5 and mycelia at pH 6.5. During bud formation, a filament ring forms just under the plasma membrane at the mother cell-bud junction at roughly the time of evagination. A filament ring also forms in mycelium-forming cells, but it appears later than in a budding cell and it is positioned along the elongating mycelium, on the average 2 microns from the mother cell-mycelium junction. Sections of filament rings in early and late budding cells and in mycelia appear similar. Each contains approximately 11 to 12 filaments equidistant from one another and closely associated with the plasma membrane. In both budding and mycelium-forming cells, the filament ring disappears when the primary septum grows inward. The close temporal and spatial association of the filament ring and the subsequent chitin-containing septum suggests a role for the filament ring in septum formation. In addition, a close temporal correlation is demonstrated between filament ring formation and the time at which cells become committed to bud formation at pH 4.5 and mycelium formation at pH 6.5. The temporal and spatial differences in filament ring formation between the two growth forms also suggest a simple model for the positioning of the filament ring.