Early Phase Cultures Research Articles

Ceramics of the early-middle Neolithic of Vodla V settlement in east Karelia

The purpose of this study is to consider the contacts between the carriers of Sperrings ceramics, pit-comb ceramics and Kargopol type ceramics on the example of materials from the early Middle Neolithic settlement of Vodla V. Sperrings ceramics dates back to the beginning of the IV millennium BC, as indicated by ornamentation, as well as traces of the influence of Karelian pit-comb ceramics and Kargopol ware. The complex of pit-comb ceramics can be attributed to the early phase of culture, however, based on new AMS definitions, the time of its existence can be dated to a later time the second quarter the middle of the IV millennium BC. The presence of vessels in the complex of Karelian pit-comb ceramics with traces of the influence of the Kargopol ornamental tradition on it also indicates the penetration of the territory of Eastern Karelia in the Neolithic of alien groups of the population with Kargopol type dishes, which is an independent type of ceramics based on its large number and presence in twenty settlements of Vodlozero Lake. In general, questions remain open about the territorial boundaries of the distribution of Karelian pit-comb ceramics and Kargopol ware, their interactions and chronological frameworks, which require processing and research of a huge block of ceramic materials of Eastern Karelia and the Southeastern Prionezhye.

Increased biomass and lipid production by continuous cultivation of Nannochloropsis salina transformant overexpressing a bHLH transcription factor.

Microalgae are promising feedstocks for sustainable and eco‐friendly production of biomaterials, which can be improved by genetic engineering. It is also necessary to optimize the processes to produce biomaterials from engineered microalgae. We previously reported that genetic improvements of an industrial microalga Nannochloropsis salina by overexpressing a basic helix‐loop‐helix transcription factor (NsbHLH2). These transformants showed an improved growth and lipid production particularly during the early phase of culture under batch culture. However, they had faster uptake of nutrients, resulting in earlier starvation and reduced growth during the later stages. We attempted to optimize the growth and lipid production by growing one of the transformants in continuous culture with variable dilution rate and feed nitrogen concentration. Relative to wild‐type, NsbHLH2 transformant consumed more nitrate at a high dilution rate (0.5 day −1), and had greater biomass production. Subsequently, nitrogen limitation at continuous cultivation led to an increased fatty acid methyl ester production by 83.6 mg l −1 day −1. To elucidate genetic mechanisms, we identified the genes containing E‐boxes, known as binding sites for bHLH transcription factors. Among these, we selected 18 genes involved in the growth and lipid metabolism, and revealed their positive contribution to the phenotypes via quantitative real‐time polymerase chain reaction. These results provide proof‐of‐concept that NsbHLH2 can be used to produce biomass and lipids.

Whole-cell Escherichia coli lactate biosensor for monitoring mammalian cell cultures during biopharmaceutical production.

ABSTRACTMany high‐value added recombinant proteins, such as therapeutic glycoproteins, are produced using mammalian cell cultures. In order to optimize the productivity of these cultures it is important to monitor cellular metabolism, for example the utilization of nutrients and the accumulation of metabolic waste products. One metabolic waste product of interest is lactic acid (lactate), overaccumulation of which can decrease cellular growth and protein production. Current methods for the detection of lactate are limited in terms of cost, sensitivity, and robustness. Therefore, we developed a whole‐cell Escherichia coli lactate biosensor based on the lldPRD operon and successfully used it to monitor lactate concentration in mammalian cell cultures. Using real samples and analytical validation we demonstrate that our biosensor can be used for absolute quantification of metabolites in complex samples with high accuracy, sensitivity, and robustness. Importantly, our whole‐cell biosensor was able to detect lactate at concentrations more than two orders of magnitude lower than the industry standard method, making it useful for monitoring lactate concentrations in early phase culture. Given the importance of lactate in a variety of both industrial and clinical contexts we anticipate that our whole‐cell biosensor can be used to address a range of interesting biological questions. It also serves as a blueprint for how to capitalize on the wealth of genetic operons for metabolite sensing available in nature for the development of other whole‐cell biosensors. Biotechnol. Bioeng. 2017;114: 1290–1300. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

Cell reproductive patterns in the green alga Pseudokirchneriella subcapitata (=Selenastrum capricornutum) and their variations under exposure to the typical toxicants potassium dichromate and 3,5-DCP.

Pseudokirchneriella subcapitata is a sickle-shaped freshwater green microalga that is normally found in unicellular form. Currently, it is the best known and most frequently used species of ecotoxicological bioindicator because of its high growth rate and sensitivity to toxicants. However, despite this organism’s, our knowledge of its cell biology—for example, the patterns of nuclear and cytoplasmic division in the mitotic stage—is limited. Although it has been reported that P. subcapitata proliferates by popularity forming four daughter cells (autospores) through multiple fission after two nuclear divisions, here, we report two additional reproductive patterns by which two autospores are formed by binary fission (“two-autospore type”) and eight autospores are formed by multiple fission (“eight-autospore type”). Moreover, we found that cell reproductive patterns differed markedly with the culture conditions or with exposure to either of two typical toxicants, potassium dichromate (K2Cr2O7) and 3,5-dichlorophenol (3,5-DCP). The eight-autospore type occurred at the highest frequency in the early phase of culture, but it disappeared under 3,5-DCP at 2.0 mg/L. Under 0.3 mg/L K2CrO7 (Cr(VI)) the eight-autospore type took substantially longer to appear than in control culture. The two-autospore type occurred only in the late phase of culture. To our knowledge, this is the first detailed evaluation of the reproductive patterns of P. subcapitata, which changed dramatically in the presence of toxicants. These findings suggest that observation of the reproductive patterns of P. subcapitata will help to elucidate different cell reactions to toxicants.

Factors Affecting Primary Cultures of Abalone Haliotis discus hannai Ovary-dissociated Cells and General Culture Aspects

We investigated factors affecting primary cultures of Pacific abalone Haliotis discus hannai ovary-dissociated cells to identify general aspects of their early-phase culture. Ninety-seven cell populations derived from 30 individuals were cultured in different media with varying compositions of medium supplements, and initial attachment, subculture, and survival for <TEX>${\geq}10$</TEX> weeks were assessed according to medium composition and individual. We also examined the time required for subculture and the rate of cell death according to both culturing period and passage number within 10 weeks. A lack of fetal bovine serum (FBS) and hemolymph significantly inhibited the growth of cultured cells, while we detected no significant effect of medium composition on initial cell attachment. Through data reallocation, with the omission of data from cell populations cultured in FBS-free and hemolymph-free media, we showed that growth inhibition was also affected by individual differences among the abalones used. During the culture, we observed four different types of cell morphology. Moreover, considerable time was required for subculture-18.4 and 19.5 days for first and second subcultures, respectively-and cell death did not occur within 30 days or for passage 0. Our results will provide valuable information for developing universal cell culturing guidelines in abalone species and suggest the feasibility of culturing abalone ovary-dissociated cells.

Tularemia: potential role of cytopathology in differential diagnosis of cervical lymphadenitis: Multicenter experience in 53 cases and literature review

Tularemia is a zoonosis caused by Francisella tularensis. Tularemia outbreaks occurred in Central Anatolia during 2009 and 2011. We evaluated the clinical characteristics and cytomorphologies of fine needle aspirations (FNAs) from cervical lymph nodes in serologically confirmed tularemia cases. To our knowledge, this is the first large series concerning FNA morphology of Tularemia. FNA smears of 53 patients of the 290, diagnosed by microagglutination tests and PCR, were evaluated at three Pathology centers. FNAs were performed by cytopathologists or ear-nose-throat surgeons. Of all patients, 17 had also lymph node resections. FNAs showed the presence of suppuration and abscess. Rare epithelioid histiocytes and granulomas, seldom phagocytosed bacilli-like microorganisms were observed. On histopathology; granulomas, necrosis, and suppurative inflammation extending extracapsular areas were seen. Tularemia is endemic in certain areas of the Northern Hemisphere. The benefit from cytopathology is limited and cytological suspicion should be confirmed by serology. However FNA cytology is helpful in differential diagnosis of tularemia and other diseases presented with suppurative, granulomatous cervical lymphadenitis. It is also useful in providing the material for PCR and culture in early phase when the serology is negative and the treatment is more effective.

In vivo augmentation of NK cell activity by methanol extract of cultured cambial meristematic cells of mountain wild ginseng (P4393)

Abstract Although mountain wild ginseng (MWG, Panax ginseng C.A.Meyer) has been regarded to be more effective than cultivated ginseng, very few data have been accumulated until now because of its limited availability and expensiveness. Some of us established a technique of isolation and culture of innately undifferentiated cambial meristematic cells from MWG as an attractive alternative. Using methanol extract of cultured MWG cambium-derived cells (MEGC), we here investigated in vivo immunomodulatory effect in a mouse model. Mice were intraperitoneally given MEGC (200 mg/Kg) and adaptive immunity was unaffected by MEGC treatment. Without Ag immunization, splenic NK cell activity in MEGC-treated mice was significantly higher when compared to controls. However, number of NK1.1+ cells and surface expression of activation/ inhibitory receptors and activation markers were unaltered by MEGC. Instead, RT-PCR data clearly demonstrated that MEGC treatment increases constitutive expression of Granzyme B and perforin in fresh spleen cells. When spleen cells were cocultured with Yac-1 in the presence of IL-2, Granzyme B and IFNγ levels were also higher in MEGC-treated group. However, these effects were achieved solely at early phase of culture (&amp;lt; day 1) and lower concentration of IL-2. Collectively, MEGC augments in vivo NK cell activity through up-regulated constitutive expression of cytolytic mediators and data suggest that ginseng may act at early phase of NK cell response.

Culturing on Wharton's Jelly Extract Delays Mesenchymal Stem Cell Senescence through p53 and p16INK4a/pRb Pathways

Mesenchymal stem cells (MSCs) hold great therapeutic potential. However, MSCs undergo replication senescence during the in vitro expansion process. Wharton's jelly from the human umbilical cord harbors a large number of MSCs. In this study, we hypothesized that Wharton's jelly would be beneficial for in vitro expansion of MSCs. Wharton's jelly extract (WJEs), which is mainly composed of extracellular matrix and cytokines, was prepared as coating substrate. Human MSCs were isolated and cultured on WJE-coated plates. Although the proliferation capacity of cells was not augmented by WJE in early phase culture, adynamic growth in late-phase culture was clearly reduced, suggesting that the replicative senescence of MSCs was efficiently slowed by WJE. This was confirmed by β-galactosidase staining and telomere length measurements of MSCs in late-phase culture. In addition, the decreased differentiation ability of MSCs after long-term culture was largely ameliorated by WJE. Reactive oxygen species (ROS), p53, and p16INK4a/pRb expression increased with passaging. Analysis at the molecular level revealed that WJE-based culture efficiently suppressed the enhancement of intracellular ROS, p53, and p16INK4a/pRb in MSCs. These data demonstrated that WJE provided an ideal microenvironment for MSCs culture expansion in vitro preserved MSC properties by delaying MSCs senescence, and allowed large numbers of MSCs to be obtained for basic research and clinical therapies.

Perfused culture of gingival fibroblasts in a degradable/polar/hydrophobic/ionic polyurethane (D-PHI) scaffold leads to enhanced proliferation and metabolic activity

Periodontal diseases cause the breakdown of the tooth-supporting gingival tissue. In treatments aimed at gingival tissue regeneration, tissue engineering is preferred over the common treatments such as scaling. Perfused (dynamic) culture has been shown to increase cell growth in tissue-engineered scaffolds. Since gingival tissues are highly vascularized, it was desired to investigate the influence of perfusion on the function of human gingival fibroblasts (HGF) when cultured in a degradable/polar/hydrophobic/ionic polyurethane scaffold during the early culture phase (4weeks) of engineering gingival tissues. It was observed that the growth of HGF was continuous over 28days in dynamic culture (3-fold increase, p<0.05), while it was reduced after 14days in static culture (i.e. no flow condition). Cell metabolic activity, as measured by a WST-1 assay, and total protein production show that HGF were in different metabolic states in the dynamic vs. static cultures. Observations from scanning electron microscopy and type I collagen (Col I) production measured by Western blotting suggest that medium perfusion significantly promoted collagen production in HGF after the first 4weeks of culture (p<0.05). The different proliferative and metabolic states for HGF in the perfused scaffolds suggest a different cell phenotype which may favour tissue regeneration.

A collagen-coated surface enables quantitative evaluation of morphological behaviors of rabbit chondrocytes relating to cell differentiation in an early culture phase

A surface with collagen type I coating was used as a tool mimicking three dimensional environment for studying the behaviors of rabbit chondrocyte aggregates found in an early culture phase. The morphology of cell aggregates was evaluated quantitatively in terms of a morphology-relating parameter of specific branching factor, Z s, determined under varied culture conditions. Branching in aggregates increased along with proceeding of culture from 5 to 6 days when seeded at a lower density of X 0 = 1.0 × 10 3 cells/cm 2, giving Z s = 0.24 tips/cell at 6 days, the value of which was 1.7 times than that obtained from the culture with a higher seeding density of X 0 = 1.0 × 10 4 cells/cm 2 ( Z s = 0.14 tips/cell). This value significantly reduced to Z s = 0.13 tips/cell in the culture with X 0 = 1.0 × 10 3 cells/cm 2 by using the conditioned medium prepared from the foregone culture with X 0 = 1.0 × 10 4 cells/cm 2, inducing the morphological architecture and collagen type II production of aggregated cells with the similarities to those in the standard culture seeded at 1.0 × 10 4 cells/cm 2. The mRNA expressions of differentiation-marker genes (collagen types I and II) were confirmed to be regulated in accordance with the morphological analyses as well as the extracellular matrix formation of aggregates in the both cultures conducted with the standard and conditioned media. The present study suggested that soluble factor(s) secreted from the chondrocytes at the higher seeding density modulates the cell aggregation and the state of chondrogenic differentiation through encouraging cell–cell communications.

Influence of microgroove dimension on cell behavior of human gingival fibroblasts cultured on titanium substrata

The purpose of this study was to determine the dimension of surface microgrooves on titanium (Ti) substrata that shows the greatest positive influence on characterizing specific cell behavior of cultured human gingival fibroblasts. Commercially pure Ti disks with surface microgrooves of monotonous 3.5 mm in depth and respective 15, 30, and 60 microm in width were fabricated using photolithography and used as the culture substrata in the three experimental groups in this study (TiD15, TiD30, and TiD60 groups), whereas the smooth Ti disk was used as the control substrata (smooth Ti group). Human gingival fibroblasts were cultured on the four groups of Ti substrata on successive timelines. Cell behaviors, such as adhesion, morphology,viability and proliferation, and gene expression were analyzed and compared between all groups using crystal violet stain, scanning electron microscopy (SEM), XTT assay, and reverse transcriptase-polymerase chain reaction, respectively. SEM demonstrated that cells were able to readily descend into the microgrooves of TiD30 at the early phase of culture. Cells on the ridge edges or in groove corners were spindle shaped with abundant filopodia formation toward the acid-etched surface inside the microgrooves, thus mimicked the cell shape in three-dimensional (3D) nanoenvironment. TiD15 significantly increased the cell viability and proliferation compared with the smooth Ti substrata after 72 h of culture. Up-regulation of fibronectin(FN) and alpha5 integrin genes was noted in cells cultured on TiD15 and TiD30. Gene expression pattern specific to the cells in 3D-matrix culture, such as down-regulation of alpha-smooth muscle actin gene along with up-regulation of FN and p21 genes, was pronounced in cells cultured on TiD30. This study indicates that surface microgrooves of both 15 and 30 microm in widt hand a monotonous 3.5 microm in depth on Ti substrata increase various cell behaviors of cultured human gingival fibroblasts.

Effect of Transforming Growth Factor-β1 on Morphological Characteristics Relating to Migration and Differentiation of Rabbit Chondrocytes Cultured in Collagen Gels

The influence of transforming growth factor-beta1 (TGFbeta1) on the behavior of rabbit chondrocytes embedded in collagen gels was examined in terms of cell migration and consequent architecture of cell aggregation. In a low-seeding density culture (X(0)=2.0 x 10(5) cells/cm(3)) TGFbeta1 (0-10.0 ng/ml) was added and observed during a 14-d culture period. Stereoscopic observation was performed on 5 d employing the morphology-related parameter of sphericity (S(c)) for individual cells in the gels. The frequency of migrating cells with S(c) less than 0.95 increased in a dose-dependent manner in response to TGFbeta1. Moreover, the frequency of migrating cells in the culture with 10.0 ng/ml TGFbeta1 was 0.32, two times higher than that in the reference culture without TGFbeta1, while the frequency of dividing cells in the same culture was less than half of that in the reference culture. The histological observation of cultured gels on 14 d revealed that the starburst and loose aggregates with the spindle-shaped cells emerged in the TGFbeta1-free culture, accompanying the poor production of collagen type II by the cells. On the other hand, the spherical-shaped cells were observed in the starburst aggregates with rich excretion of collagen type II in the culture with 5.0 ng/ml TGFbeta1. Moreover, the mRNA levels of differentiation-marker genes (collagen types I and II) were regulated in accordance with the morphological analyses concerning the cell migration and aggregation in the cultures with and without TGFbeta1. From these results, it was concluded that TGFbeta1 had a culture time-dependent effect on the morphological characteristics relating to the migration and differentiation of the chondrocytes in the collagen gel-embedded cultures seeded at low density, that is, the growth factor promotes cell migration with deteriorated proliferation in the early culture phase, and accelerates the transformation of spindle-shaped cells to spherical-shaped ones in the prolonged culture.

Seeding density modulates migration and morphology of rabbit chondrocytes cultured in collagen gels

The cultures of rabbit chondrocytes embedded in collagen gels were conducted to investigate the cell behaviors and consequent architectures of cell aggregation in an early culture phase. The chondrocyte cells seeded at 1.0 x 10(5) cells/cm(3) underwent a transition to spindle-shaped morphology, and formed the loose aggregates with a starburst shape by means of possible migration and gathering. These aggregates accompanied the poor production of collagen type II, while the cells seeded at 1.6 x 10(6) cells/cm(3) exhibited active proliferation to form the dense aggregates rich in collagen type II. Stereoscopic observation was performed at 5 days to define the migrating cells in terms of a morphology-relating parameter of sphericity determined for individual cells in the gels. The frequency of migrating cells decreased with increasing seeding density, while the frequency of dividing cells showed the counter trend. The culture seeded at 1.0 x 10(5) cells/cm(3) gave the migrating cell frequency of 0.25, the value of which was 25 times higher than that at 1.6 x 10(6) cells/cm(3). In addition, the analysis of mRNA expression revealed that the chondrocyte cells seeded at 1.0 x 10(5) cells/cm(3) showed appreciable down-regulation in collagen type II relating to differentiation and up-regulation in matrix metalloproteinases relating to migration, as compared to the cells seeded at 1.6 x 10(6) cells/cm(3). These data supports the morphological analyses concerning the cell migration and aggregate formation in the cultures with varied seeding densities. It is concluded that the seeding density is an important factor to affect the cell behaviors and architecture of aggregates and thereby to modulate the quality of cultured cartilage.

Influence of diurnal hyperosmotic loading on the metabolism and matrix gene expression of a whole‐organ intervertebral disc model

It is generally agreed that the mechanical environment of intervertebral disc cells plays an important role in maintaining a balanced matrix metabolism. The precise mechanism by which the signals are transduced into the cells is poorly understood. Osmotic changes in the extracellular matrix (ECM) are thought to be involved. Current in-vitro studies on this topic are mostly short-term and show conflicting data on the reaction of disc cells subjected to osmotic changes which is partially due to the heterogenous and often substantially-reduced culture systems. The aim of the study was therefore to investigate the effects of cyclic osmotic loading for 4 weeks on metabolism and matrix gene expression in a full-organ intervertebral disc culture system. Intervertebral disc/endplate units were isolated from New Zealand White Rabbits and cultured either in iso-osmotic media (335 mosmol/kg) or were diurnally exposed for 8 hours to hyper-osmotic conditions (485 mosmol/kg). Cell viability, metabolic activity, matrix composition and matrix gene expression profile (collagen types I/II and aggrecan) were monitored using Live/Dead cell viability assay, tetrazolium reduction test (WST 8), proteoglycan and DNA quantification assays and quantitative PCR. The results show that diurnal osmotic stimulation did not have significant effects on proteoglycan content, cellularity and disc cell viability after 28 days in culture. However, hyperosmolarity caused increased cell death in the early culture phase and counteracted up-regulation of type I collagen gene expression in nucleus and annulus cells. Moreover, the initially decreased cellular dehydrogenase activity recovered with osmotic stimulation after 4 weeks and aggrecan gene down-regulation was delayed, although the latter was not significant according to our statistical criteria. In contrast, collagen type II did not respond to the osmotic changes and was down-regulated in both groups. In conclusion, diurnal hyper-osmotic stimulation of a whole-organ disc/endplate culture partially inhibits a matrix gene expression profile as encountered in degenerative disc disease and counteracts cellular metabolic hypo-activity.

Distribution and density of bacteria in subtropical flooded rice growing areas in Brazil

Since microorganisms are important components in the structure and function of water systems, the present paper reports work done to identify groups making up bacterial communities present in rice irrigation water. To this end, water samples were collected in five rice-growing areas of Rio Grande do Sul State, Brazil. The identification of the main bacterial groups found in flooded areas was carried out based on colony morphology, taking into account shape, elevation, and edges. Internal and external cell structure was characterized as cocci or rod, gram-positive or gram-negative, and spore forming or not. The results obtained regarding density and bacterial distribution in the water samples from rice growing areas of RS showed a higher abundance of morphotypes of bacterial colonies in the irrigation channels (F(1,12) = 8.74; p < 0.05) when compared to the culture plots in the five production regions, and in the final culture phase (F(1,12) = 8.86; p < 0.05). In the five rice production areas the occurrence of gram-positive and spore-forming rods was prevalent. Rods showed more abundance (F(1,26) = 15.12; p < 0.05) in the early culture phase, with a significant prevalence of gram-positive rods (F(1,26) = 25.99; p < 0.05) in all rice growing regions under investigation. The group of spore-forming bacteria was significantly more abundant (F(1,30) = 14.06; p < 0.05) when compared with the non-spore-forming ones in all investigated regions. Data showed that the irrigated rice crop affects the density and morphotype colonies in the bacteria found in the water used to flood rice-growing plots.

Transdifferentiation of Bone Marrow-Derived Cells Co-Cultured with Fetal Liver Cells into Hepatic-Like Cells without Fusion.

Several research groups have reported that bone marrow cells (BMCs) transdifferentiate into hepatocytes in rodents. However, it is yet to be studied what factors effectively trigger and sustain the transdifferentiation of BMCs to hepatocytes. In the present study, we investigated whether murine BMCs in the presence of fetal liver cells (FLCs) could differentiate into hepatic-like cells in vitro without fusion. Fractionated BMCs from C57Bl/6-TgN(ACTbEGFP)10bs mice and FLCs from B6.129S7-Gt(ROSA)26Sor mice were co-cultured at 1x105 cells and 1x106 cells in 10% FCS-containing medium supplemented with hepatocyte growth factor on laminin-coated dishes. Hepatocyte-specific markers among BMCs were detected as assessed by immunocytochemistry for albumin and reverse transcription-polymerase chain reaction (RT-PCR) for alpha-fetoprotein (AFP), albumin, and cytokeratin-19 mRNAs. We also found that Sca-1+ BMCs containing both hematopoietic stem cells and AFP-expressing cells could differentiate into hepatic-like cells and such cells were seen adherent to dish together with FLCs in the early phase of culture. Moreover, the AFP-expressing cells were found in a Sca-1+ cKit- cell fraction, which also differentiated into CD45− GFP+ albumin+ cells and proved to be positive for GFP but negative for LacZ as assessed by RT-PCR and immunocytochemistry. These results suggest that albumin+ cells developed through transdifferentiation from BMCs but not through spontaneous cell fusion between BMCs and FLCs.

Distinct effects of caudalizing factors on regional specification of embryonic stem cell-derived neural precursors

Recent embryological studies have implicated several “caudalizing factors” in the caudal specification of the central nervous system (CNS). In this study, we have examined the effects of three candidate caudalizing factors on neural precursors induced from embryonic stem (ES) cells by the stromal cell-derived inducing activity (SDIA) method. Among retinoic acid (RA), Wnt and FGF signals, RA causes the strongest level of caudalization: inducing suppression of forebrain differentiation and promotion of caudal CNS specification. Obvious suppression of the telencephalic marker Bf1 and that of the forebrain marker Otx2 occur at 2×10 −8 and 2×10 −7 M, respectively. Activation of the caudal marker genes such as Hoxb9 is observed in a dose-dependent manner over the range of 2×10 −9–2×10 −6 M. Suppression of the forebrain genes has a narrow critical period of RA response during the early culture phase. In contrast, significant induction of the caudal genes is evoked by a 1-day exposure to RA at any time between days 3 and 8. RA treatment not only induces caudal specification but also inhibits differentiation of ventral CNS tissues, particularly of floor plate cells. FGF4 induces partial caudalization while Wnt-3A exhibits weak caudalizing activities only in the presence of RA. These findings provide useful information on the proper selection of combination of signaling molecules, doses and timing for steering ES cell differentiation by caudalizing factors into caudal neural fates.

Promoted proliferation of an SOD-deficient mutant ofEscherichia coliunder oxidative stress induced by photoexcited TiO2

An SOD null mutant of Escherichia coli (IM303) and its wild-type strain (MM294) were cultivated with or without sublethal oxidative stress generated from photoexcited TiO2. Concerning maximum specific growth rate of the cells, mum, measured under various conditions, the mum value of IM303 cells increased notably in the presence of TiO2 illuminated with light (I = 12.5 W m(-2)), being about two times higher than that of the cells grown in the absence of TiO2 and light. The mum value of IM303 cells under the oxidative condition restored to a level comparable to that of wild-type MM294 cells, which coincided with the finding that the content of reactive oxygen species lowered in IM303 cells under the oxidative stress. Colony isolation was conducted to obtain the cells prevailing in the early culture phase of IM303 cells in the presence of TiO2 and light. It was found that the isolates exhibited the outgrowing properties with the increased mum values under both the conditions with and without TiO2 and light. It was also indicated that in the culture of typically selected isolate, the cells started to grow with a relatively short lag in a threonine-minus medium.

Promoted proliferation of an SOD-deficient mutant of Escherichia coli under oxidative stress induced by photoexcited TiO 2

An SOD null mutant of Escherichia coli (IM303) and its wild-type strain (MM294) were cultivated with or without sublethal oxidative stress generated from photoexcited TiO 2. Concerning maximum specific growth rate of the cells, μ m, measured under various conditions, the μ m value of IM303 cells increased notably in the presence of TiO 2 illuminated with light ( I=12.5 W m −2), being about two times higher than that of the cells grown in the absence of TiO 2 and light. The μ m value of IM303 cells under the oxidative condition restored to a level comparable to that of wild-type MM294 cells, which coincided with the finding that the content of reactive oxygen species lowered in IM303 cells under the oxidative stress. Colony isolation was conducted to obtain the cells prevailing in the early culture phase of IM303 cells in the presence of TiO 2 and light. It was found that the isolates exhibited the outgrowing properties with the increased μ m values under both the conditions with and without TiO 2 and light. It was also indicated that in the culture of typically selected isolate, the cells started to grow with a relatively short lag in a threonine-minus medium.

Activation of cell proliferation by brassinolide application in tobacco BY-2 cells: effects of brassinolide on cell multiplication, cell-cycle-related gene expression, and organellar DNA contents.

Brassinosteroids (BRs) are steroidal phytohormones that are essential for many processes in plant growth and development, such as cell expansion, vascular differentiation, and responses to stress. The effects of BRs on cell division are unclear, as attested by contradictory published results. To determine the effect of BRs on cell division, the tobacco (Nicotiana tabacum) BY-2 cell line, which is a widely-used model system in plant cell biology, was used. It was found that brassinolide (BL) promoted cell division only during the early phase of culture and in the absence of auxin (2,4-D). This promotion of cell division was confirmed by RNA gel blot analyses using cell-cycle-related gene probes. At later stages in the culturing periods of BL-supplied and 2,4-D-supplied BY-2 cells, differences in cell multiplication and cell-cycle-related gene expression were observed. Moreover, the BL-treated BY-2 cells had morphological differences from the 2,4-D-treated cells. To determine whether suppressed organellar DNA replication limited this promotion of cell division during the early culture phase, this replication was examined and it was found that BL treatment had no effect on activating organellar (plastid- and mitochondrial-) DNA synthesis. As preferential organellar DNA synthesis, which is activated by 2,4-D, is necessary during successive cell divisions in BY-2 cells, these data suggest that the mechanism of the promotion of cell division by BL treatment is distinct from that regulated by the balance of auxin and cytokinin.