Food deprivation is not a prerequisite for the amoebal to plasmodial transition in Physarum polycephalum

The transmission pattern of mitochondrial DNA (mtDNA) was studied during plasmodium formation in Physarum polycephalum. Plasmodia were generated by matings between pairs of amoebal strains carrying mtDNA molecules that were distinguishable by restriction endonuclease digestion. The transmission of mtDNA was uniparental in every case; the plasmodia always carried mtDNA with the restriction pattern of only one of the two parental types. In each mating pair, one strain consistently acted as mtDNA donor, but this strain did not always act as mtDNA donor when combined in other mating pairs. The identity of the mtDNA donor in each pair was not determined by the different types of mtDNA molecules present or by different alleles of matB or matC, two mating-type loci which regulate amoebal fusion. The results suggested that alleles of a third mating-type locus, matA, which controls zygote development, might form a hierarchy such that the mtDNA donor in any cross would be the strain of higher status. The deduced hierarchy was matA2 > matA11 > matA12 > matA1.

Epidermal growth factor (EGF) counteracts the stimulation of glycogen synthesis by insulin in hepatocytes, but it is not known whether this is due to inhibition of glycogen synthesis or to inhibition of the insulin-signalling mechanism. This study investigates the mechanisms by which EGF affects the basal rate and the insulin stimulation of glycogen synthesis. The basal rate of glycogen synthesis is higher at low than at high cell density. EGF inhibits the basal rate of glycogen synthesis at low cell density but not in confluent cultures and abolishes the difference due to density. However, EGF inhibits the stimulation of glycogen synthesis by insulin irrespective of cell density. Increasing glycogen synthesis by increasing the [glucose] does not abolish the difference in rates of glycogen synthesis due to cell density, neither does it induce responsiveness to EGF at high cell density, establishing that responsiveness to EGF is a function of cell density and not of the basal rate and that inhibition of the insulin stimulation also cannot be accounted for by the higher rate of glycogen synthesis. Cytochalasin D and phalloidin, which alter cell morphology through interactions with the microfilament cytoskeleton, mimic the cell-density-dependent inhibition of glycogen synthesis by EGF. The inhibition of glycogen synthesis by EGF and cytochalasin D is additive and cytochalasin D potentiates the inhibition of glycogen synthesis by EGF, suggesting involvement of a cytoskeletal mechanism. Exogenous phospholipase C inhibits glycogen synthesis at both low and high cell density and the inhibition at low cell density is not additive with that caused by either EGF or cytochalasin D, suggesting that these agonists inhibit glycogen synthesis through changes in Ca2+ and/or diacylglycerol. The inhibition of glycogen synthesis by EGF in the absence of insulin stimulation is blocked by neomycin, which inhibits Ca2+ release from intracellular stores but not by antagonists of protein kinase C. It was also inhibited by pertussis toxin (50%), suggesting that it may involve GTP-binding-protein-mediated release of Ca2+ from intracellular stores. The inhibition of the stimulation of glycogen synthesis by insulin was not affected by neomycin and was only marginally inhibited by pertussis toxin or guanosine 5'-O-[3-thio]triphosphate (GTP[S]). We infer from these findings that the inhibition by EGF of the basal rate of glycogen synthesis and of the insulin stimulation are mediated by different mechanisms. The latter is pertussis toxin insensitive and independent of cell density, whereas the former is expressed only at low cell density, it is potentiated by cytochalasin D and inhibited by pertussis toxin.

温度和食物水平对海月水母螅状体无性繁殖的影响

SummaryHaploid amoebae ofPhysarum polycephalummay form plasmodia by crossing, a sexual process that involves cellular and nuclear fusions, or by selfing, an asexual process in which the development of a single amoeba into a plasmodium may involve neither cellular nor nuclear fusion. Mutant strains (npf) in which selfing is suppressed were previously assigned to several functional groups on the basis of their ability to cross with one another in certain combinations. In the present study hybrid, diploid amoebae were isolated from both crossing-compatible and incompatible mixtures ofnpfmutants. The diploid amoebae from mixtures of compatible strains readily formed plasmodia by selfing, but selfing was suppressed in the diploids from incompatible mixtures. Thus the crossing tests betweennpfmutants may be viewed as complementation tests: their results reflect the differing selfing abilities of the hybrid, diploid amoebae that formed in each mixture. Genetical and environmental factors affecting the efficiency of formation of diploid amoebae were studied, and the diploids were shown to be stable during repeated subcultures. Although diploid amoebae carrying complementingnpfmutations readily formed plasmodia by selfing at 26 δC, they could be cultured without plasmodium formation at 30 δC, a temperature that also inhibited selfing of the haploidnpf+strains. Ways are discussed of exploiting this combination of properties in a general procedure for isolating and testing diploids for dominance and complementation of amoebal mutations inP. polycephalum.

The transformation of uninucleate amoebae of Physarum polycephalum into multinucleate plasmodia is under the control of a multiallelic mating-type locus, matA. Plasmodium formation usually occurs only when amoebal fusion brings together two different matA alleles within a single cell; such plasmodium formation is termed crossing. Mutations (gadA) in the matA locus permit haploid amoebae to self, that is to form plasmodia in clonal cultures without amoebal fusion. By constructing diploid amoebae heterozygous for gadA alleles, it was shown that the mutant alleles gadA5 and gadA111 were each dominant to gadA +. Non-selfing revertants of gadA mutants may be generated as a result of further mutations (npf) within the matA locus. Sixty-one revertants of this type were studied, derived from three matA3 gadA mutants. Some revertants were able to cross with a matA3 gadA + tester strain. These ‘class I’ revertants failed to cross with one another or with similar revertants of two matA2 gadA mutants; they appear all to lack a single gene function (npfC +) that is necessary in plasmodium development. Other revertants failed to cross with the matA3 gadA + tester. These ‘class II’ revertants also failed to cross with one another, but showed several different patterns of crossing when mixed with other strains. The behaviour of some of the class II revertants suggests that they lack a further gene function (npfB +) necessary in plasmodium development. The gadA mutations may affect a cis-acting regulator of npfB +.

Important considerations for cell therapy manufacturing of mesenchymal stem cell

Seagrass meadows are often important habitats for newly recruited juvenile fishes. Although substantial effort has gone into documenting patterns of association of fishes with attributes of seagrass beds, experimental investigations of why fish use seagrass habitats are rare. We performed two short-term manipulative field experiments to test (1) the effects of food supply on growth and densities of fish, and (2) effects of predation on the density and size distribution of fish recruits, and how this varies among habitat types. Experiments were conducted in Galveston Bay, Texas, and we focused on the common estuarine fish, pinfish Lagodon rhomboides. In the first experiment, replicate artifical seagrass and sand plots were either supplemented with food or left as controls. Recruitment of pinfish was significantly greater to seagrass than sand habitats; however, we detected no effect of food supplementation on the abundance of recruits in either habitat. Pinfish recruits in artifical seagrass grew at a significantly faster rate than those in sand habitats, and fish supplemented with food exhibited a greater growth rate than controls in both sand and artifical grass habitats. In our second experiment, we provided artificial seagrass and sand habitats with and without predator access. Predator access was manipulated with cages, and two-sided cages served as controls. Recruitment was significantly greater to the cage versus cage-control treatment, and this effect did not vary between habitats. In addition, the standard length of pinfish recruits was significantly larger in the predator access than in the predator exclusion treatment, suggesting size-selective predation on smaller settlers or density-dependent growth. Our results indicate that the impact of predation on pinfish recruits is equivalent in both sand and vegetated habitats, and thus differential predation does not explain the higher recruitment of pinfish to vegetated than to nonvegetated habitats. Since predators may disproportionately affect smaller fish, and a limited food resource appears to be more effectively utilized by fish in vegetated than in unvegetated habitats, we hypothesize that pinfish recruits may select vegetated habitats because high growth rates allow them to achieve a size that is relatively safe from predation more quickly.

For insect parasitoids, knowledge of their flight capability is essential for a general understanding of the relationship with their hosts. For instance, flight capacity might partly determine their efficacy as biological control agents. Ibalia leucospoides Hochenwarch (Hymenoptera: Ibaliidae) is a solitary, pro-ovigenic parasitoid of the woodwasp, Sirex noctilio Boidin (Hymenoptera, Siricidae), an economically important pest of softwood forestation. This study explores the flight capacities of I. leucospoides females and assesses the effects of a sugar-rich food supply and crowding on female flight performance, by using computer-linked flight mills. The present study shows: (1) a high variability in flight potential of I. leucospoides females, (2) no effects of food supply and grouping on wasp flight (flight distance and speed), (3) a significant effect of body size and wing loading on flight performance and, (4) a significant body mass loss during the flight dependent on the total distance flown. The lack of effect of food on a highly energy-demanding activity as flight may be related to the life-history traits and nutritional strategies of this parasitoid. The relevance of these observations for the use of I. leucospoides as a biological control agent is discussed.

Desensitization of cultured muscle cells to isoproterenol

Influence of cell density on gene transfection and the underlying mechanism remain unclear. In this study, micropatterned surfaces are prepared to control cell density, cell morphology, and cell–cell interactions to investigate how and why cell density affects gene transfection of mesenchymal stem cells. Cell density is controlled by adjusting the ratio of cell adhesive area to nonadhesive area. Gene transfection is dependent on cell density. A moderate cell density is beneficial for gene transfection, while low (2.5 ± 0.7 × 103 cells cm−2) and high (112.6 ± 9.6 × 103 cells cm−2) cell densities have negative effects on transfection. Influence of cell density on gene transfection is strongly correlated with its influence on cellular uptake capacity and DNA synthesis. High cellular uptake and DNA synthesis promote gene transfection. Variations in cell morphology and cell–cell interaction levels of populations with different cell densities could explain the influence of cell density. Large cell size (low cell density) and strong cell–cell interaction (high cell density) could increase gene transfection. Cell protrusion of large cells could slightly promote gene transfection. Balance of these factors is important for maximal gene transfection. The results should be useful for understanding the effect of cell density on gene transfection.

Normal cells in culture respond to cell density by altering their proliferation rates and their pattern of protein expression. Primary avian tendon (PAT) cells are a case in point where procollagen production increases approximately 10-fold at high cell density while proliferation almost ceases. In an earlier report focusing on the cell density regulation of procollagen expression, the signaling mechanism communicating the presence of other cells was shown to have the characteristics of a loosely bound component of the cell layer. Extending these studies to the cell density regulation of proliferation, the cell density signal (CDS) was again shown to be altered by medium agitation, stimulating cell division. Agitation, however, was only disruptive to cell signaling when there was a high ratio of medium to cells. When sufficient cells were present, agitation was less effective. Therefore, the CDS controlling procollagen production and the CDS controlling the inhibition of growth seemed to be linked because the signaling mechanism is disrupted in a parallel manner by agitation. However, the proliferative response of PAT cells is more complex in that there is also a positive influence at moderate cell density (> 2 x 10(4) cells/cm2) on the rate of cell division. As a consequence, PAT cells would not proliferate into an area of low cell density, but within the same dish would rapidly fill an area of moderate density. PAT cells were capable of filling a gap between high cell density areas if the gap was less than 2 mm. Medium agitation also affected cells at low cell density in a different manner. It was inhibitory if all the cells were at low cell density but it was stimulatory if the cells at low cell density were in close proximity to cells at high cell density. In addition, medium conditioned by agitation over cells at a high cell density would stimulate cells at low cell density to divide and grow out into low cell density regions. Using the growth-promoting activity of the conditioned medium as an assay, this component of the CDS was shown to have unique characteristics: heat, pH, dithiothreitol (DTT) stable; tris ion and protease sensitive. By gel exclusion chromatography it was larger than 100 kDa. But after DTT treatment its mobility shifted to < 30 kDa while retaining activity.

We tested specific laminin (LN) isoforms for their ability to serve as substrata for maintaining mouse embryonic stem (ES) cells pluripotent in vitro in the absence of leukemia inhibitory factor or any other differentiation inhibitors or feeder cells. Recombinant human LN-511 alone was sufficient to enable self-renewal of mouse ES cells for up to 169 days (31 passages). Cells cultured on LN-511 maintained expression of pluripotency markers, such as Oct4, Sox2, Tert, UTF1, and Nanog, during the entire period, and cells cultured for 95 days (17 passages) were used to generate chimeric mice. LN-332 enabled ES cells proliferation but not pluripotency. In contrast, under the same conditions LN-111, Matrigel, and gelatin caused rapid differentiation, whereas LN-411 and poly-d-lysine did not support survival. ES cells formed a thin monolayer on LN-511 that differed strikingly from typical dense cluster ES cell morphology. However, expression of pluripotency markers was not affected by morphological changes. The effect was achieved at low ES cell density (<200 cell/mm(2)). The ability of LN-511 and LN-332 to support ES cell proliferation correlated with increased cell contact area with those adhesive substrata. ES cells interacted with LN-511 via beta1-integrins, mostly alpha6beta1 and alphaVbeta1. This is the first demonstration that certain extracellular matrix molecules can support ES cell self-renewal in the absence of differentiation inhibitors and at low cell density. The results suggest that recombinant laminin isoforms can provide a basis for defined surface coating systems for feeder-free maintenance of undifferentiated mammalian ES cells in vitro. Disclosure of potential conflicts of interest is found at the end of this article.

Reversions of mouse adenocarcinoma (MAC/10T3) cells from fibroblastic, high-tumorigenic to epithelioid, low-tumorigenic phenotype was obtained by either seeding MAC/10T3 cells at low cell density or by exposing them to varying concentrations of IQ-1 or IQ-2 compounds. Clonal isolation was carried out by the ring isolation technique, and the various cloned revertant types were characterized by their cell morphology, saturation density, and tumorigenicity. Most of the revertants isolated 11 to 15 days after seeding at low cell density eventually returned to the fibroblastic, high-tumorigenic (HT) phenotype when serially passed at high cell density. In contrast, 25 to 36.3% of the slow-growing revertants isolated between 21 and 28 days after seeding at low cell density maintained the parental epithelioid morphology when subsequently seeded at high cell density. The observation of a possible temporal relationship between the low density seeding and the occurrence of stable revertants, and the observed differential cellular proliferation at low or high cell density, may suggest that reversion from a less tumorigenic phenotype to a highly tumorigenic state may involve a multistep process with a stringent requirement for active cell cycle.

Hypoxia-Induced Autophagy Promotes EGFR Loss in Specific Cell Contexts, Which Leads to Cell Death and Enhanced Radiosensitivity

Diabetes is accompanied by an increased autofluorescence of the cornea, probably because of accumulation of advanced glycation end products (AGEs). The pathogenic mechanism is still unknown. This study aimed to quantify differences in corneal cell densities between diabetic patients and healthy controls. The left cornea of 15 patients with non-insulin-dependent diabetes mellitus (NIDDM) with level of retinopathy 20 according to the Early Treatment of Diabetic Retinopathy Study (ETDRS) and of 15 healthy controls were examined by noninvasive in vivo confocal microscopy in an observational prospective study. The cell densities in 6 corneal layers were determined along the optical axis of the cornea by using stereologic methods. The average cell density per unit area in the superficial and basal epithelium and the endothelial layer was 725 +/- 171, 5950 +/- 653, and 2690 +/- 302 cells/mm in controls and 815 +/- 260, 5060 +/- 301, and 2660 +/- 364 cells/mm in diabetic patients. The cell density per unit volume in the anterior, mid-, and posterior stroma was 26,300 +/- 4090, 19,390 +/- 3120, and 25,700 +/- 3260 cells/mm in controls and 27,560 +/- 3880, 21,930 +/- 2110, and 25,790 +/- 3090 cells/mm in patients with diabetes. In both groups, the density in the midstroma was significantly lower than in both the anterior stroma and the posterior stroma (P < 0.02). The cell density in the basal layer of diabetic patients was significantly lower than in healthy controls (-15.0%, P < 0.0004). In the other layers, no significant differences between both groups (P > 0.07) were observed. The lower basal cell density found in patients with diabetes may result from a combination of different mechanisms including decreased innervation at the subbasal nerve plexus, basement membrane alterations, and higher turnover rate in basal epithelial cells. The lower cell density in the midstroma of diabetic patients and healthy controls may be attributed in part to differences in oxygen concentration in the stromal layers (depths). Changes in cellular density did not seem to be responsible for the increased autofluorescence in diabetes.