Discrete Colonies Research Articles

Surface-aminated electrospun nanofibers enhance adhesion and expansion of human umbilical cord blood hematopoietic stem/progenitor cells

Interaction between hematopoietic stem/progenitor cells (HSPCs) and their extra cellular matrix components is an integral part of the signaling control for HSPC survival, proliferation and differentiation. We hypothesized that both substrate topographical cues and biochemical cues could act synergistically with cytokine supplementation to improve ex vivo expansion of HSPCs. In this study, we compared the ex vivo expansion of human umbilical cord blood CD34(+) cells on unmodified, hydroxylated, carboxylated and aminated nanofibers and films. Results from 10-day expansion cultures showed that aminated nanofiber mesh and film were most efficient in supporting the expansion of the CD34(+)CD45(+) cells (195-fold and 178-fold, respectively), as compared to tissue culture polystyrene (50-fold, p<0.05). In particular, aminated nanofiber meshes supported a higher degree of cell adhesion and percentage of HSPCs, as compared to aminated films. SEM imaging revealed the discrete colonies of cells proliferating and interacting with the aminated nanofibers. This study highlights the potential of a biomaterials approach to influence the proliferation and differentiation of HSPCs ex vivo.

Structure and development of Nostoc strands in Leiosporoceros dussii (Anthocerotophyta): a novel symbiosis in land plants

The presence of Nostoc in longitudinally oriented schizogenous canals is a feature that separates Leiosporoceros from all other hornworts and represents a novel symbiotic arrangement in land plants. In surface view, Nostoc canals are visible as elongated, dichotomously branched blue-green strands. All other hornworts develop numerous discrete globose colonies through continuous production of mucilage clefts as avenues for multiple invasions within a single thallus. To elucidate the anatomy and development of the unusual Nostoc strands in Leiosporoceros, we examined sporeling development in culture and the structure of strands in field-collected plants using light and electron microscopy. Rosette-like sporelings have mucilage clefts scattered along swollen apices. All field specimens were strap-shaped, contained Nostoc, and lacked mucilage clefts. Nostoc strands are located in the center of the thallus and develop behind the apical cell by separation of the middle lamella between apical derivatives. Strands elongate and branch in synchrony with apical growth, and thus only a single invasion is required for strand production. Two distinct ultrastructural morphotypes in the collections suggest nonspecificity of Nostoc. We speculate that Nostoc enters the thallus in the sporeling stage through mucilage clefts, and once colonies are established, cleft production ceases.

Widespread gene flow and high genetic variability in populations of water volesArvicola terrestrisin patchy habitats

Theory predicts that the impact of gene flow on the genetic structure of populations in patchy habitats depends on its scale and the demographic attributes of demes (e.g. local colony sizes and timing of reproduction), but empirical evidence is scarce. We inferred the impact of gene flow on genetic structure among populations of water voles Arvicola terrestris that differed in average colony sizes, population turnover and degree of patchiness. Colonies typically consisted of few reproducing adults and several juveniles. Twelve polymorphic microsatellite DNA loci were examined. Levels of individual genetic variability in all areas were high (H(O) = 0.69-0.78). Assignments of juveniles to parents revealed frequent dispersal over long distances. The populations showed negative F(IS) values among juveniles, F(IS) values around zero among adults, high F(ST) values among colonies for juveniles, and moderate, often insignificant, F(ST) values for parents. We inferred that excess heterozygosity within colonies reflected the few individuals dispersing from a large area to form discrete breeding colonies. Thus pre-breeding dispersal followed by rapid reproduction results in a seasonal increase in differentiation due to local family groups. Genetic variation was as high in low-density populations in patchy habitats as in populations in continuous habitats used for comparison. In contrast to most theoretical predictions, we found that populations living in patchy habitats can maintain high levels of genetic variability when only a few adults contribute to breeding in each colony, when the variance of reproductive success among colonies is likely to be low, and when dispersal between colonies exceeds nearest-neighbour distances.

Microbiology of Fresh-cut Mangoes Prepared from Fruit Sanitised in Hot Chlorinated Water

Whole mangoes ( Mangifera indica, cv. Chok Anun) were washed in water with or without chlorine (100 mg total chlorine/L) at 11.7 ºC and 50 ºC before slicing and packaging. Microbiological analysis of the whole fruit showed that the stem scar contained larger populations of microorganisms than the skin. Washing reduced populations at both sites and the effect was enhanced by the addition of chlorine and heating of the wash water. None of the treatments prevented transfer of contaminants to the flesh during slicing, and evidence of spoilage in the form of discrete fungal colonies was observed in samples stored for 1 week at 5 ºC. Several species of bacteria, yeast and mould were recovered from packaged slices, but yeast and moulds accounted for the bulk of the spoilage of microflora. Barriers to the growth of these microorganisms may be necessary to reduce the risk of microbial spoilage of mango slices manufactured under commercial conditions.

Habitat assessment by parasitoids: mechanisms for patch use behavior

Animals foraging for patchily distributed resources may optimize their foraging decisions concerning the patches they encounter, provided that they base these decisions on reliable information about the profitability of the habitat as a whole. Females of the parasitoid Lysiphlebus testaceipes exploit aphid hosts, which typically aggregate in discrete colonies. We show here how betweencolony travel time and the number of aphids in previously visited colonies affect parasitoid foraging behavior. We first assumed that parasitoids use travel time and previous colony size to estimate a mean rate of fitness gain in the habitat and derived quantitative predictions concerning the effect of these two variables on patch residence time and patch-leaving rate of attack. We then tested these theoretical predictions in laboratory experiments in which female parasitoids were allowed to visit two successive colonies. As predicted, the observed residence time in the second colony increased with increasing travel time and decreasing size of the first colony. Patch-leaving rate of attack decreased with increasing travel time but was not affected by previous colony size. These results suggest that parasitoids use these two variables to assess habitat quality. However, discrepancies between the data obtained and quantitative predictions show that the effect of travel time on patch use may be more complex than assumed in our model. Key words: learning, Lysiphlebus testaceipes, marginal value theorem, optimal foraging theory, patch size, travel time. [Behav Ecol]

AMD3100 and G-CSF Mobilize Angiogenic Cells into the Blood of Healthy Donors.

There is evidence that angiogenic cells exist in the blood that are able to home to sites of ischemia and induce angiogenesis. Angiogenic cell populations identified in human blood include endothelial progenitor cells (EPC) and circulating angiogenic monocytes (CAM). Under basal conditions, however, the number of angiogenic cells in the circulation is small, potentially limiting their delivery to sites of ischemia and subsequent stimulation of angiogenesis. To circumvent this limitation, animal studies have shown that angiogenic cells can be mobilized into blood by certain cytokines; whether a similar mobilization of angiogenic cells can be achieved in humans is not clear. Herein, we report the results of a clinical trial that examined the ability of G-CSF and AMD3100 to mobilize EPC and CAM into the blood. Donors were treated with a single injection of AMD3100 (5mg/kg) after which pheresis was performed. One week later, donors were treated with G-CSF (250microg/kg/d x 5 days) and pheresed. Blood was collected at baseline (prior to the initiation of the mobilizing regimen) and after treatment with AMD3100 or G-CSF (at the time of peak HSC mobilization). In addition, pheresis product was collected after mobilization by AMD3100 or G-CSF. Mononuclear cells (MNC) were isolated from the blood products and cultured under angiogenic conditions. EPC were identified by the formation of discrete colonies of endothelial cells on days 14-28. A novel method to quantify CAM was developed to avoid the pitfalls of counting adherent cells in culture. Briefly, adherent cells were recovered from the culture, counted, and analyzed by flow cytometry. CAM were identified as CD45+ CD14+ CD31+ cells. The angiogenic potential of the CAM was assessed by their transplantation into NOD/SCID/b2M−/− mice following surgical induction of hindlimb ischemia. Indeed, reperfusion was significantly enhanced in mice that received CAM compared to control mice [ratio±SEM) of ischemic to non-ischemic limb perfusion: 0.47±0.05 (saline treated); 0.78±0.09 (CAM transplant); p<.001]. To date, a total of 9 patients have been analyzed. AMD3100 resulted in a 9.2-fold increase in CAM in the blood [CAM/ml peripheral blood±SEM: 3.5x105±0.09 (AMD3100) vs. 0.4x105±0.01 (baseline); p<.05]. Likewise, treatment with G-CSF resulted in a 12.3-fold increase in CAM (4.5x105±0.21; p<.05). Both AMD3100 and G-CSF also mobilized EPC into the blood [EPC/mL peripheral blood±SEM: 0.53±0.22 (AMD3100); 0.98±0.24 (G-CSF); vs. 0.05±0.01 (baseline); p<.05 for both AMD3100 and G-CSF compared with baseline]. Interestingly, data suggests that the G-CSF-mobilized CAM may have an enhanced capacity to stimulate angiogenesis in the hindlimb ischemia model [the ratio of ischemic to non-ischemic limb perfusion±SEM: 0.47±0.05 (saline treated); 0.64+0.11 (G-CSF) p<.001; 0.58±0.06 (AMD3100); p=NS]. Importantly, CAM and EPC were readily recovered from the pheresis product after both AMD3100 and G-CSF treatment. Collectively these data show that treatment with AMD3100 or G-CSF mobilizes a significant number of CAM and EPC into the peripheral circulation. These mobilized angiogenic cells have the capacity to stimulate angiogenesis in vivo. Finally, CAM and EPC can be efficiently harvested by leukopheresis, providing a method to obtain large numbers of circulating angiogenic cells for subsequent clinical use.±

Embryonic Erythropoiesis and Definitive Hematopoiesis from Human Embryonic Stem Cells Is Regulated by Cytokines Controlling HSC Growth.

Human embryonic stem cells (hESC) provide an invaluable tool for studying the earliest events in human hematopoietic stem cell (HSC) development. We describe novel protocols for the efficient, step-wise differentiation of hESC to embryonic (primitive) erythroid cells and definitive erythro-myeloid cells from embryoid bodies (EB) in semi-solid and liquid cultures. EB cells were re-cultured in semisolid cultures with a cocktail of hematopoietic growth factors at different time points using a modified EB differentiation protocol, and hematopoietic differentiation was analyzed in vitro.The initiation of hematopoiesis, in this model, begins during the first week of EB differentiation. with the formation of primitive macrophages and CD31+/VE-cadherin+ hemato-endothelial clusters that “bud off” primitive embryonic hemoglobin-expressing erythroblasts and multi-potential blast colonies. These clusters ultimately form organized yolk-sac-like structures which produce a loosely adherent primitive hematopoietic cells. After 7–9 days of EB differentiation (prior to CD45 expression), primitive nucleated erythroblast colonies arise and are characterized by a “brilliant red” hemoglobinization under phase microscopy, positivity for embryonic/fetal hemoglobins by Kleihauer-Betke, qRT-PCR assays for epsilon/zeta/gamma chain expression, and a CD71+/glycophorin A+ phenotype. Simultaneously, discrete blast colonies are also shown to develop into mixed multipotential colonies containing secondary erythro-myeloid blast cells, primitive erythroblasts, and macrophages; suggesting a common progenitor for the discrete embryonic phenotypes observed at this stage. Following this first wave of primitive hematopoiesis, definitive CD45+-expressing colony-forming cells (CFC) can be generated from EB cells differentiated for 10–15 days with the sequential appearance of BFU-E, CFU-E, GM-CFC, and multi-lineage CFC. A kinetic expression analysis using qRT-PCR methods, revealed that the first wave of embryonic hematopoiesis at 6–9 days of EB development directly coincides with expression of SCL/TAL1, AML1, GATA1, and GATA2, while the onset of definitive hematopoiesis at 9–15 days directly correlates with increased EB expression of CD34, CD31, CD41, c-myb, and cdx4.In this model, primitive hematopoiesis in EB cells proceeds in the absence of exogenously added growth factors. However, supplementing EB differentiation cultures with FLT3-ligand, KIT-ligand, and THROMBOPOIETIN (FTK), dramatically enhances the number of primitive erythroblast, and multi-lineage blast CFC, as well as the definitive BFU-E, CFU-E, and multi-potent mixed CFC. The kinetics of colony formation for both primitive and definitive CFC is unaffected by FTK supplementation. Moreover, blast cell colonies from EB cells differentiated in the presence of FTK were more potent than those generated without FTK. These blast colonies differentiate into mixed, multi-lineage CD45+/CD13+/CD41+/CD71+/glycophorin A+-expressing colonies that contain both primitive nucleated embryonic hemoglobin-expressing erythroblasts, and definitive mature beta-globin-expressing erythroid cells, neutrophils, monocytes/macrophages, and megakaryocytic precursors. This hESC model reveals the putative existence of a common human progenitor for both embryonic-type and definitive hematopoietic cells, and that cytokines known to expand/self-renew definitive HSC may potentially regulate this differentiation process.

Geographic variation and acoustic structure of the underwater vocalization of harbor seal (Phoca vitulina) in Norway, Sweden and Scotland

The male harbor seal (Phoca vitulina) produces broadband nonharmonic vocalizations underwater during the breeding season. In total, 120 vocalizations from six colonies were analyzed to provide a description of the acoustic structure and for the presence of geographic variation. The complex harbor seal vocalizations may be described by how the frequency bandwidth varies over time. An algorithm that identifies the boundaries between noise and signal from digital spectrograms was developed in order to extract a frequency bandwidth contour. The contours were used as inputs for multivariate analysis. The vocalizations' sound types (e.g., pulsed sound, whistle, and broadband nonharmonic sound) were determined by comparing the vocalizations' spectrographic representations with sound waves produced by known sound sources. Comparison between colonies revealed differences in the frequency contours, as well as some geographical variation in use of sound types. The vocal differences may reflect a limited exchange of individuals between the six colonies due to long distances and strong site fidelity. Geographically different vocal repertoires have potential for identifying discrete breeding colonies of harbor seals, but more information is needed on the nature and extent of early movements of young, the degree of learning, and the stability of the vocal repertoire. A characteristic feature of many vocalizations in this study was the presence of tonal-like introductory phrases that fit into the categories pulsed sound and whistles. The functions of these phrases are unknown but may be important in distance perception and localization of the sound source. The potential behavioral consequences of the observed variability may be indicative of adaptations to different environmental properties influencing determination of distance and direction and plausible different male mating tactics.

Two novel species of tetrodotoxin-producing bacteria isolated from toxic marine puffer fishes

Out of eight dominant discrete bacterial colonies isolated and purified from the toxic marine puffer fishes collected in Hong Kong waters, two novel species of non-sporing, non-acid-fast and chemoorganotrophic bacteria capable of producing tetrodotoxin (TTX, a potent non-protein neurotoxin), as well as one previously reported and confirmed TTX-producing bacterium. They were identified as Microbacterium arabinogalactanolyticum, Serratia marcescens and Vibrio alginolyticus, respectively, all of which are widely distributed in soils, sewage or marine environments. Each bacterial isolate (500 ml broth medium cultured in darkness without aeration for 10 days at 25 °C) could produce an amount of toxicity, after extraction and purification, ranging from 78.3 to 105.3 mouse units (MU) in 500 ml of broth medium by mouse bioassay. The principal toxic component in the bacterial cultures was determined to be TTX by thin layer chromatography and mass spectrometry.

High-throughput imaging of bacterial colonies grown on filter plates with application to serum bactericidal assays

The ability to accurately enumerate viable bacteria has applications in antibiotic screening assays, toxicology testing, and serological assays for functional antibodies. An impediment to high-throughput bacterial assays is the requirement to grow bacteria as individual colonies on semisolid media containing agar. We have now developed a method for growth, staining, and counting of bacterial colonies in 96-well filter plates. A unique feature of the method is that colony size is inversely proportional to the number of colonies in each well, presumably due to nutrient depletion. As a result, as many as 300 colony-forming units (cfu) can be detected as discrete colonies within a single assay well. The resulting colonies can be counted automatically using an imaging system originally developed for ELISPOT assays. The method has been applied to the measurement of serum bactericidal activity (SBA) in human sera.

Isolation and cultivation of filamentous bacteria implicated in activated sludge bulking

Filamentous bacteria have long been associated with activated sludge bulking and foaming and are known to be the main cause of this problem. Chemical control methods such as chlorination and the use of hydrogen peroxide have been, and still are, used to cure bulking and foaming but are only effective as interim measures. More detailed understanding of the physiology and biochemistry of filamentous bacteria is still required for effective long-term control of bulking and foaming. Isolation and cultivation of filamentous bacteria in pure culture have shown promise as methods to gaining better understanding of bulking and foaming. The aim of this project was, therefore, to assess different techniques for effective isolation and cultivation of filamentous bacteria in pure culture. Activated sludge samples from Durban and surrounding areas were screened microscopically to identify constituent filamentous bacterial populations. Samples with varieties of different filamentous bacteria were subjected to various floc break-up procedures (nonidet surfactant treatment, sonication and cellulase hydrolysis) in conjunction with physical separation (centrifugation) to separate filamentous bacteria from floc-forming bacteria. Both treated and untreated samples were serially diluted and plated onto a variety of different solid media, whereafter discrete bacterial colonies were isolated and screened microscopically for filamentous morphology. Cellulase hydrolysis proved unsuccessful for filament isolation while direct inoculation, nonidet pretreatment and sonication resulted in the isolation of five different filaments, one via sonication and two each via the other methods. The filaments were provisionally identified as Sphaerotilus natans, Microthrix parvicella, Type 1863, Type 0092 and Haliscomenobacter hydrossis. WaterSA Vol.29(4) 2003: 405-410

Modeling of a microbial growth experiment with bioclogging in a two-dimensional saturated porous media flow field

A model was developed simulating reactive transport in groundwater including bioclogging. Results from a bioclogging experiment in a flow cell with a two-dimensional flow field were used as a data base to verify the simulation results of the model. Simulations were performed using three different hydraulic conductivity vs. porosity relations published in literature; two relations derived from pore network simulations assuming the biomass to grow in discrete colonies and as a biofilm, respectively, and a third relation, which did not include pore connectivity in more than one dimension. Best agreement with the experimental data was achieved using a hydraulic conductivity vs. porosity relation derived from pore network simulation assuming the biomass to grow in colonies. The relation derived from pore network simulations assuming biomass to grow as a biofilm was unable to reproduce the experimental data when realistic parameter values were employed. With the third relation the clogging ability of the biomass was strongly underestimated. These findings indicate that the porous medium needs to be treated as a multi-dimensional medium already on the pore scale, and that biomass growth different than in a biofilm must be considered to get an appropriate description of bioclogging.

Influence of Food on Numbers Breeding, Colony Size and Fidelity to Localities of Swift Terns in South Africa’s Western Cape, 1987-2000

During 1987-2000, the numbers of Swift Terns (Sterna bergii) breeding in South Africa’s Western Cape varied between 1,449 and 5,668 pairs, distributed at 13 localities. Numbers were significantly related to the combined biomass of Anchovy (Engraulis capensis) and Sardine (Sardinops sagax), two of the main food items. In years when food was scarce, numbers breeding decreased by up to 67%. The sizes of breeding aggregations of Swift Terns were significantly related to the combined biomass of Anchovy and Sardine. Numbers of Swift Terns breeding at a locality ranged from 1-4,192 pairs, with an average of 750. Numbers breeding in discrete colonies ranged from 1-3,000 pairs, with an average of 513. Swift Terns showed low fidelity to breeding areas, 61% of which were used just once. This is attributed to their requirement to breed near to fish prey, and its variable distribution from year to year. Breeding by other seabirds probably often indicated safe sites for nesting. Swift Terns bred by themselves in 17%, with Hartlaub’s Gulls (Larus hartlaubii) in 77%, and with Crowned Cormorants (Phalacrocorax coronatus) in 18% of breeding attempts.

Influence of Microbial Growth on Hydraulic Properties of Pore Networks

From laboratory experiments it is known that bacterial biomass is able to influence the hydraulic properties of saturated porous media, an effect called bioclogging. To interpret the observations of these experiments and to predict possible bioclogging effects on the field scale it is necessary to use transport models, which are able to include bioclogging. For these models it is necessary to know the relation between the amount of biomass and the hydraulic conductivity of the porous medium. Usually these relations were determined using bundles of parallel pore channels and do not account for interconnections between the pores in more than one dimension. The present study uses two-dimensional pore network models to study the effects of bioclogging on the pore scale. Numerical simulations were done for two different scenarios of the growth of biomass in the pores. Scenario 1 assumes microbial growth in discrete colonies clogging particular pores completely. Scenario 2 assumes microbial growth as a biofilm growing on the wall of each pore. In both scenarios the hydraulic conductivity was reduced by at least two orders of magnitude, but for the colony scenario much less biomass was needed to get a maximal clogging effect and a better agreement with previously published experimental data could be found. For both scenarios it was shown that heterogeneous pore networks could be clogged with less biomass than more homogeneous ones.

Low nutrient medium and spiral-plate system for enumerating heterotrophic bacteria in soil

Identifying appropriate media to quickly and accurately enumerate bacteria in soil has been a continuing problem in microbiology research. Our objective was to determine the accuracy of the spiral plating technique to assess bacteria population in soil samples and its compatibility with low and high nutrient media. Colony numbers and the area covered by spreading organisms at 48, 72, and 144 hr were compared among four culture media (Standard Methods Agar [SMA] at 1%, 10%, and 100% strength, and Modified Egg Albumen Agar [MAA]) using two contrasting soils. The correlation between the spiral and the spread plating methods on MAA was tested using 17 soil samples. MAA gave the same or higher colony counts compared to full strength or diluted SMA. Colony size was larger on MAA than on 1% or 10% SMA. Confluent colonies that overgrew diluted and full strength SMA plates were largely absent from MAA plates. There were no differences between colony counts from spiral plating and spread plating methods using MAA. Colony numbers (log CFU g−1 dry soil) from both methods on several soils were strongly related (R2=0.97, n= 17, p<0.01) and followed the relationship y= 1.008x - 0.125 where y represents the spread plate method and x the spiral plate method. Use of MAA media with the spiral plating technique provides automation of bacteria enumeration without sacrificing either numerical counts or the appearance of discrete colonies.

Water vole in the Scottish uplands: distribution patterns of disturbed and pristine populations ahead and behind the American mink invasion front

AbstractThe invasion of Britain by American mink has had a catastrophic impact on water vole populations. We surveyed and live‐trapped water voles over 2 years in eight c. 25 km2 blocks in the upland of Scotland behind and ahead of the mink invasion front. Water voles had a similar distribution in the Grampian Mountains of north‐east Scotland, on the edge of the invasion front, and in the Assynt area of north‐west Sutherland well beyond the front. Water voles occurred in small, discrete colonies. Median nearest‐neighbour distance between colonies was 0.6‐0.7 km in both areas. Colonies experienced a high degree of turnover with extinction and colonization being commonplace and only a fraction of suitable sites were occupied at a given time. High dispersal rates connecting numerous (&gt; 30) colonies over large areas (&gt; 25 km2) enable water voles to persist in such circumstances. Synchronized fluctuations in occupancy not caused by mink also occurred at the regional scales of the Grampian Mountains and Assynt areas. Localized mink invasions have fragmented a previously continuous metapopulation into smaller clusters and this may indirectly affect the likely persistence of water vole colonies not directly exposed to predation by mink.

Dose-response relationship for radiation-induced mutations at micro- and minisatellite loci in human somatic cells in culture

Purpose : The study was designed to determine the dose-response relationship for radiation induction of mutations at mini- and microsatellite loci in human somatic cells. Mutations induced by graded doses of gamma-irradiation were quantified by screening clones derived from single irradiated cells for micro- and minisatellite alterations following irradiation with 1, 2 or 3 Gy. Materials and methods : After irradiation, the moderately radioresistant glioma cell line UVW was seeded at low density into Petri dishes to allow formation of discrete colonies, 100 of which were examined at each dose. All the cells within a colony were presumed to have arisen from a single irradiated cell. Radiation-induced microsatellite alterations were determined at 16 different loci, by PCR amplification and visualization on polyacrylamide gels. Minisatellite alterations were identified at four different minisatellite loci by restriction enzyme digestion and Southern blotting. Results : A dose-response curve for mutation frequency was obtained by analysis of 100 clones, yielding a minisatellite mutation rate of 5.5 X 10 -3 mutations/locus/Gy/cell and a microsatellite mutation rate of 8.75 X 10 -4 mutations/locus/Gy/cell. At microsatellite loci, alterations were predominantly simple loss or gain of repeat units and loss of heterozygosity (LOH). The mutations in minisatellite loci resulted predominantly in LOH and variation in repeat number. The background instability at each locus was determined by analysis of non-irradiated clones. Only 2% and 1% of the micro- and minisatellite loci respectively showed altered bands. Conclusions : This is the first report of a dose-response relationship for radiation-induced micro- and minisatellite mutations in human somatic cells. Described is a sensitive method for analysis of low-dose radiation mutagenesis in somatic cells that may prove to be a useful tool for radiation protection and dosimetry.

Relation of the slow growth phenotype to neoplastic transformation: possible significance for human cancer.

Deletions are widely distributed over the genome in the most frequently occurring human cancers and are the most abundant genetic lesion found there. Deletions are highly correlated with the slow growth phenotype of mutated animal and human cells and result in chromosomal transposition when the retained ends are joined. Transpositions are only a minor source of mutation in rapidly multiplying bacteria but are a major cause of mutations in stationary bacteria. The NIH 3T3 line of mouse cells undergoes neoplastic transformation during prolonged incubation in a stationary state and expresses the slow growth phenotype on serial subculture at low density, suggesting a relation between transformation and chromosomal deletions. To further explore the relation between neoplastic transformation and the slow growth phenotype as a surrogate for deletions, two sublines of the NIH 3T3 cells with differing competence for transformation were serially subcultured in the stationary state at confluence and tested at each subculture for transformation and growth rate. Cell death in a fraction of the population and a heritable slowdown in proliferation of most of the survivors became increasingly pronounced with successive rounds of confluence. The reduction in growth rate was not proportional to the degree of transformation of the cultures, but all of the transformed cultures were slow growers at low density. All of the discrete colonies from cloning transformed cultures developed at a lower initial rate than control colonies under optimal conditions for growth, but they continued to grow at later stages, forming multilayered colonies under conditions that inhibited the further growth of the control colonies. The results suggest that prolonged incubation of NIH 3T3 cells in the stationary state results in growth-impairing deletions over a wide range of sites in the genome, but more restricted subsets of such lesions are responsible for neoplastic transformation. These findings provide dynamic, functional support in culture for the histopathological evidence that the quiescent state of cells associated with atrophy and fibrosis plays a significant role in the origin of some cancers in experimental animals and human beings.

Kinetics of osteoprogenitor proliferation and osteoblast differentiation in vitro

Fetal rat calvaria cells plated at very low density generate discrete colonies, some of which are bone colonies (nodules) from individual osteoprogenitors that divide and differentiate. We have analyzed the relationship between cell proliferation and acquisition of tissue-specific differentiation markers in bone colonies followed individually from the original single cell to the fully mineralized state. The size distribution of fully formed nodules is unimodal, suggesting that the coupling between proliferation and differentiation of osteoprogenitor cells is governed by a stochastic element, but distributed around an optimum, corresponding to the peak colony size/division potential. Kinetic analysis of colony growth showed that osteoprogenitors undergo 9-10 population doublings before the appearance of the first morphologically differentiated osteoblasts in the developing colony. Double immunolabeling showed that these proliferating cells express a gradient of bone markers, from proliferative alkaline phosphatase-negative cells at the periphery of colonies, to postmitotic, osteocalcin-producing osteoblasts at the centers. An inverse relationship exists between cell division and expression of osteocalcin, the latter being restricted to late-stage, BrdU-negative osteoblasts, while the expression of all other markers is acquired before the cessation of proliferation, but not concomitantly. Bone sialoprotein expression is biphasic, detectable in some of the early, alkaline phosphatase-negative cells, and again later in both late preosteoblast (BrdU-positive) and osteoblast (BrdU-negative, osteocalcin-positive) cells. In late-stage, heavily mineralized nodules, staining for osteocalcin and bone sialoprotein is not detectable in the oldest/most mature cells. Our observations support the view that the bone nodule "tissue-like" structure, originating from a single osteoprogenitor and finally encompassing mineralized matrix production, recapitulates successive stages of the osteoblast differentiation pathway, in a proliferation/maturation sequence. Understanding the complexity of the proliferation/differentiation kinetics that occurs within bone nodules will aid in the qualitative and/or quantitative interpretation of tissue-specific marker expression during osteoblastic differentiation.

Natal origins of migratory monarch butterflies at wintering colonies in Mexico: new isotopic evidence.

Each year, millions of monarch butterflies from eastern North America migrate to overwinter in 10-13 discrete colonies located in the Oyamel forests of central Mexico. For decades efforts to track monarch migration have relied on observations and tag-recapture methods, culminating with the discovery of the wintering colonies in 1975. Monarch tag returns from Mexico, however, are few and primarily from two accessible colonies, and therefore tag-recapture techniques have not quantified natal origins or distinctiveness among monarch populations at wintering sites. Such information would be invaluable in the conservation of the monarch and its migration phenomenon since the wintering sites currently are threatened by habitat alteration. Here we show that stable hydrogen (deltaD) and carbon (delta13C) isotope ratios of wintering monarchs can be used to evaluate natal origins on the summer breeding range. Stable-hydrogen and carbon isotopic values of 597 wintering monarchs from 13 wintering roost sites were compared with isotopic patterns measured in individuals at natal sites across their breeding range over a single migration cycle. We determined that all monarch wintering colonies were composed of individuals originating mainly from the Midwest, United States, thereby providing evidence for a panmictic model of wintering colony composition. However, two colonies showed more northerly origins, suggesting possible priority colonies for conservation efforts.