High Density Culture Conditions Research Articles

Effects of Lactobacillus plantarum and Bacillus subtilis on growth, immunity and intestinal flora of largemouth bass(Micropterus salmoides)

Probiotics play a crucial role in largemouth bass aquaculture, primarily in enhancing water quality, preventing fish diseases, promoting largemouth bass growth, and boosting immunity in high-density culture conditions. The experiment was actualized by adding Lactobacillus plantarum, Bacillus subtilis and a mixture of probiotic (L. plantarum and B. subtilis 1:1) in the culture water of largemouth bass for 42 days, respectively, to explore the effect of the above two probiotics on the growth performance and immunity ability of largemouth bass (Micropterus salmoides).Results: At the level of phylum, the relative abundance of enteric flora in Tenericutes of largemouth bass in L. plantarum added group was significantly higher than that in control group (p < 0.05). Conversely, the relative abundance of Proteobacteria was significantly lower than that of control group (p < 0.05). Similarly, at the genus level, the relative abundance of Mycoplasma and Leifsonia was significantly higher than that of control group (p < 0.05), while the relative abundance of Aeromonas was significantly lower in the L. plantarum added group compared to the control group (p < 0.05). Compared with the control group, there were no significant differences in phylum level in the B. subtilis added group (p < 0.05), and the relative abundance of Plesiomonas was significantly increased (p < 0.05), the relative abundance of Aeromonas was significantly decreased at genus level (p < 0.05). The relative abundance of Plesiomonas and Parabacteroides in the mixed probiotic treatment group was significantly increased (p < 0.05), the relative abundance of Aeromonas was significantly decreased at the level of genus (p < 0.05). The metabolites of carboxylic acid and its derivatives, organic oxygen compound, glycerophospholipid, lip acyl and other categories in enteric tissues of all treatment groups had significant changes (p < 0.05), enteric bacterial secondary metabolites such as SCFAs and 1-Phosphatidyl-D-myo-inositol were significantly increased (p < 0.05). The results showed that the addition of L. plantarum and B. subtilis in water could promote the growth of largemouth bass and increase its nonspecific immunity by enhancing the stability of enteric flora, increasing efficiency of enteric digestion and absorption and promoting the anti-inflammatory and immune ability of the body through secondary metabolites of enteric microbe.

Transcriptomics of Lactobacillus paracasei: metabolism patterns and cellular responses under high-density culture conditions.

Lactobacillus paracasei has significant potential for development and application in the environmental field, particularly in addressing malodor pollution. This study aims to investigate the cellular response of L. paracasei B1 under high-density culture conditions. The selected strain has previously shown effective deodorizing and bacteriostatic abilities. Transcriptomics techniques are employed to dissect the nutrient metabolism pattern of L. paracasei B1 and its response mechanism under environmental stress. The study characterizes the functions of key differentially expressed genes during growth before and after optimizing the culture conditions. The optimization of fermentation culture conditions provides a suitable growth environment for L. paracasei B1, inducing an enhancement of its phosphotransferase system for sugar source uptake and maintaining high levels of glycolysis and pyruvate metabolism. Consequently, the strain is able to grow and multiply rapidly. Under acid stress conditions, glycolysis and pyruvate metabolism are inhibited, and L. paracasei B1 generates additional energy through aerobic respiration to meet the energy demand. The two-component system and quorum sensing play roles in the response and regulation of L. paracasei B1 to adverse environments. The strain mitigates oxygen stress damage through glutathione metabolism, cysteine and methionine metabolism, base excision repair, and purine and pyrimidine metabolism. Additionally, the strain enhances lysine synthesis, the alanine, aspartate, and glutamate metabolic pathways, and relies on the ABC transport system to accumulate amino acid-compatible solutes to counteract acid stress and osmotic stress during pH regulation. These findings establish a theoretical basis for the further development and application of L. paracasei B1 for its productive properties.

Differentiation of Human Induced Pluripotent Stem Cells (iPSCs)-derived Mesenchymal Progenitors into Chondrocytes.

Induced pluripotent stem cells (iPSCs) generated from human sources are valuable tools for studying skeletal development and diseases, as well as for potential use in regenerative medicine for skeletal tissues such as articular cartilage. To successfully differentiate human iPSCs into functional chondrocytes, it is essential to establish efficient and reproducible strategies that closely mimic the physiological chondrogenic differentiation process. Here, we describe a simple and efficient protocol for differentiation of human iPSCs into chondrocytes via generation of an intermediate population of mesenchymal progenitors. These methodologies include step-by-step procedures for mesenchymal derivation, induction of chondrogenic differentiation, and evaluation of the chondrogenic marker gene expression. In this protocol, we describe the detailed procedure for successful derivation of mesenchymal progenitor population from human iPSCs, which are then differentiated into chondrocytes using high-density culture conditions by stimulating with bone morphogenetic protein-2 (BMP-2) or transforming growth factor beta-3 (TGFβ-3). The differentiated iPSCs exhibit temporal expression of cartilage genes and accumulation of a cartilaginous extracellular matrix in vitro, indicating successful chondrogenic differentiation. These detailed methodologies help effective differentiation of human iPSCs into the chondrogenic lineage to obtain functional chondrocytes, which hold great promise for modeling skeletal development and disease, as well as for potential use in regenerative medicine for cell-based therapy for cartilage regeneration. Key features • Differentiation of human iPSCs into chondrocytes using 3D culture methods. • Uses mesenchymal progenitors as an intermediate for differentiation into chondrocytes.

YAP Promotes Cell Proliferation and Stemness Maintenance of Porcine Muscle Stem Cells under High-Density Condition.

Muscle stem cells (MuSCs) isolated ex vivo are essential original cells to produce cultured meat. Currently, one of the main obstacles for cultured meat production derives from the limited capacity of large-scale amplification of MuSCs, especially under high-density culture condition. Here, we show that at higher cell densities, proliferation and differentiation capacities of porcine MuSCs are impaired. We investigate the roles of Hippo-YAP signaling, which is important regulators in response to cell contact inhibition. Interestingly, abundant but not functional YAP proteins are accumulated in MuSCs seeded at high density. When treated with lysophosphatidic acid (LPA), the activator of YAP, porcine MuSCs exhibit increased proliferation and elevated differentiation potential compared with control cells. Moreover, constitutively active YAP with deactivated phosphorylation sites, but not intact YAP, promotes cell proliferation and stemness maintenance of MuSCs. Together, we reveal a potential molecular target that enables massive MuSCs expansion for large-scale cultured meat production under high-density condition.

Tryptophan supplementation helps totoaba (Totoaba macdonaldi) juveniles to regain homeostasis in high-density culture conditions.

High-density culture brings with it chronic stress situations that affect fish welfare. In order to evaluate the effect of tryptophan (Trp) levels on the response to stress, Totoaba macdonaldi juveniles were stocked at low (13.5kgm-3) and high (27.0kgm-3) densities (32.5 and 56.4kgm-3, respectively, at the end of the experiment) in 100-L tanks and fed for 63days with experimental diets containing different Trp levels: control diet CD0.42 (0.42%) and three supplemented diets with 0.99, 1.55 and 2.19% (0.99Trp, 1.55Trp and 2.19Trp, respectively) (three tanks × density × diet). The high-density stocking fed with CD0.42 diets showed significantly increased blood parameters. Trp decreased catalase (CAT) activity in low- and high-density stocking, while the superoxide dismutase (SOD) activity showed no difference. Serotonin (5-hydroxytryptamine, 5-HT) content decreased, and the serotonin turnover ratio (5-HIAA:5-HT) increased in the brains of fish fed with the CD0.42 diet. Indeed, Trp-supplemented diets helped to restore homeostasis in high-density growth conditions as evaluated by the hematological and plasma parameters as well as the serotonergic activity. When the fish were provided a diet containing moderate Trp levels, plasma cortisol increased under high-density conditions. However, no differences were observed among stock densities when totoaba were fed with the 2.19Trp diet. Notably, survival was unaffected by both Trp or densities, but weight gain (WG) decreased with the dietary Trp levels in the high density culture. In sum, Trp supplementation decreased the parameter values linked to stress response on totoaba juveniles cultured at high stock densities.

High-Efficiency Lentiviral Transduction of Human CD34+ Cells in High-Density Culture with Poloxamer and Prostaglandin E2

Hematopoietic stem cell (HSC) gene therapy is curative for various hereditary diseases; however, high-efficiency transduction in HSCs remains crucial to improve the prospects for hemoglobinopathies. We previously optimized lentiviral transduction in human CD34+ cells with serum-free medium containing minimal cytokines, allowing efficient transduction (∼50%) and robust xenograft engraftment. In this study, we further improved lentiviral transduction in human CD34+ cells. High-density culture conditions (4e6/mL) resulted in ∼5-fold more efficient transduction in CD34+ cells (p < 0.01) compared with standard cell density (1e5/mL). After co-culturing vector-exposed CD34+ cells with non-transduced CD34+ cells, high-density culture conditions enhanced lentiviral gene marking in the non-transduced population (p < 0.01) compared with low-density conditions, suggesting that increasing cell-to-cell contact allows more efficient transduction. Two adjuvants, poloxamer 407 (100 μg/mL) and prostaglandin E2 (10 μM), were added to high-density CD34+ cells, resulting in ∼4-fold more efficient transduction (p < 0.01) without significant toxicity compared with no adjuvant control. In summary, we developed a highly efficient lentiviral transduction method in high-density CD34+ cell culture with poloxamer 407 and prostaglandin E2, allowing overall ∼10-fold improvement in transduction efficiency and consistently achieving more than 90% transduction and an average vector copy number of ∼10. Our optimized transduction method should improve gene therapy approaches using lentiviral vectors targeting HSCs.

Abstract 921: Pericellular-interactions promote malignant progression of glioblastoma via novel NOTCH-1/HIF-1α pathway

Abstract Malignant tumors generally develop hypoxic condition in the core region of tumor mass, which induces stabilization of hypoxia inducible factor-1 alpha (HIF-1α) by preventing proteasome-dependent degradation. HIF-1α has been shown to regulate various oncogenes for malignant progression of many types of tumors including glioblastoma multiforme (GBM). In the present study, we showed that cell-to-cell interactions at high density can induce malignant transformation of tumor cells by activation of HIF-1α even under normoxic condition. To delineate the biological effect of cell culture density, we maintained cancer cells in different density. Four types of GBM cell lines (U251-MG, LN215-MG, U373-MG, and CRT-MG) were used to examine the oncogenic phenotypes of cancer cells cultured in different densities. Cells cultured in a high-density condition showed higher rate of proliferation and migration activity and higher resistance to temozolomide, an anti-cancer drug commonly used for GBM treatment. We found that the pericellular interactions stimulate the cleavage of notch-1 intracellular domain (NICD), and the cleaved NICD protects the HIF-1α from hydroxylation and subsequent proteasomal degradation, resulting in nuclear translocation of HIF-1α. Collectively, we suggest that the cell-cell interactions in high-density culture conditions induce NICD-mediated HIF-1α activation, which leads to malignant phenotypic changes. Therefore, treatment of gamma-secretase inhibitor (GSI), which can block NOTCH-1 signaling pathway, in combination with TMZ could be a promising therapeutic strategy in TMZ-resistant tumor cells. This research was supported by a grant of the Korea Health Technology R&amp;D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health &amp; Welfare, Republic of Korea (grant number : HI14C0042) Citation Format: Eunsoo Kim, Jungwhoi Lee, Seung-Wook Ryu, Chulhee Choi, Kyungsun Choi. Pericellular-interactions promote malignant progression of glioblastoma via novel NOTCH-1/HIF-1α pathway. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 921.

Differentiation of Human Induced Pluripotent Stem Cells to Chondrocytes.

Human induced pluripotent stem (iPS) cells are relevant tools for modeling human skeletal development and disease, and represent a promising source of patient-specific cells for the regeneration of skeletal tissue, such as articular cartilage. Devising efficient and reproducible strategies, which closely mimic the physiological chondrogenic differentiation process, will be necessary to generate functional chondrocytes from human iPS cells. Our previous study demonstrated the generation of chondrogenically committed human iPS cells via the enrichment of a mesenchymal-like progenitor population, application of appropriate high-density culture conditions, and stimulation with bone morphogenetic protein-2 (Bmp-2). The differentiated iPS cells showed temporal expression of cartilage genes and the accumulation of a cartilaginous extracellular matrix in vitro. In this chapter, we provide detailed methodologies for the differentiation of human iPS cells to the chondrogenic lineage and describe protocols for the analysis of chondrogenic differentiation.

Derivation and long-term culture of an embryonic stem cell-like line from zebrafish blastomeres under feeder-free condition.

Existing zebrafish embryonic stem (ES) cell lines are derived and maintained using feeder layers. We describe here the derivation and long-term culture of an ES cell-like line derived from zebrafish blastomeres without the use of feeder cells. This line, designated as ZES1, has been maintained for more than 800 days in defined Dulbecco's modified Eagle's medium supplemented with fetal bovine serum, zebrafish embryo extract, trout serum, and human basic fibroblast growth factor. ZES1 cells possessed a morphology typical of ES cells, being round or polygonal in shape with a large nucleus and sparse cytoplasm and were mostly diploid. The cells formed individual colonies consisting of tightly packed cells that stained positively for alkaline phosphatase. ZES1 cells also formed embryoid bodies when transferred onto uncoated wells. The pluripotent nature of ZES1 cells was confirmed when they could be induced to differentiate in vitro into several cell types, through low- or high-density culture conditions. Treatment with retinoic acid also induced the differentiation of ZES1 cells into primarily neuronal cells. Using immunostaining and real-time polymerase chain reaction, we showed that Sox2, a known pluripotent marker in mammalian ES cells, was also present in ZES1 cells. Chimera experiments revealed that fluorescent-labeled ZES1 cells microinjected into zebrafish blastulas participated in the formation of all three germ layers. Using GFP-labeled ZES1 cells, chimera germline transmission was also demonstrated at the F1 generation. In conclusion, ZES1 cells possess both in vitro and in vivo pluripotency characteristics, indicating that nonmammalian ES cells can be readily derived and maintained for a long term under feeder-free culture conditions.

Growth, Metabolic and Physiological Response of Juvenile Cheraxquadricarinatus Fed Different Available Nutritional Substrates

There are many factors in intensive culture conditions which vary the physiological response of organisms in culture affecting performance such as growth, metabolic levels in blood, respiratory metabolism and the inflection point of dissolved oxygen concentration. The study was conducted under high density culture conditions with multitrophic systems. Results show that the species can tolerate intensive culture conditions without affecting their growth and survival and has the ability to leverage multiple food sources. This work also shows that Cheraxquadricarinatus has the ability to maintain a low routine metabolic rate, is highly tolerant to limited dissolved oxygen conditions and thus uses energy efficiently. The metabolic rate in juvenile C. quadricarinatus was 0.07 ± 0.003 mg O2/g/h while the critical level of oxygenconcentration, when the organism passes from regulator to conformer is 0.483 ± 0.002 mg O2/L. This is significantly lower than previously reported for cultured decapod species.

Ebp1 activates podoplanin expression and contributes to oral tumorigenesis

Podoplanin is highly expressed in human cancers. However, mechanisms regulating podoplanin expression remain elusive. Here we show that podoplanin promotes tumorigenesis of oral squamous cell carcinoma (OSCC) and precancerous cells both in vitro and in vivo, and the ErbB3-binding protein-1 (Ebp1) can be activated in oral tumorigenesis and can serve as a transcriptional activator to drive podoplanin expression in the malignant progression. Most of the OSCC cell lines have no detectable podoplanin protein in low-density cultures. However, the protein becomes detectable in high-density cultures and is required for in-vivo tumor formation of OSCC and oral premalignancies. In a high-density culture condition, podoplanin expression can be triggered at both mRNA and protein levels. In this condition, we showed that Ebp1 is upregulated, translocated from the cytoplasm to the nucleus and binds to the podoplanin promoter to result in a dramatic increase of podoplanin mRNA and protein. Ebp1 downregulation significantly reduced podoplanin expression levels in OSCC cells with a decreased anchorage-dependent growth, invasion and wound healing. Conversely, Ebp1 overexpression enhanced these malignant features through podoplanin upregulation both in vitro and in vivo. In 81 patients with oral premalignant lesions, we found that Ebp1 expression is strongly related to OSCC development. We conclude that Ebp1 has a key role in the upregulation of podoplanin and may contribute to oral tumorigenesis.

Lysyl Oxidase Is Induced by Cell Density-Mediated Cell Cycle Suppression via RB-E2F1-HIF-1α Axis

Remodeling of the matrix surrounding tumor cells plays a crucial role in the development and maintenance of cancer. Lysyl oxidase (LOX), a matrix remodeling factor, is induced by HIF-1α under hypoxic conditions and associated with tumor growth and metastasis. Here, we report that high cell density induces HIF-1α expression under normoxic condition, resulting in the promotion of LOX expression. This phenomenon was observed in the retinoblastoma tumor suppressor (RB)-proficient breast cancer cells but not in RB-deficient cells. In RB-proficient cancer cells, the cell cycle regulator E2F1 was down-regulated and cell cycle progression was inhibited at high density culture condition. Knockdown of E2F1 stabilized HIF-1α and promoted LOX expression, while knockdown of both E2F1 and HIF-1α prevented the up-regulation of LOX. These findings suggest that elevated cell density enhances cell cycle arrest and matrix remodeling via RB-E2F1-HIF-1α axis.

Gene Expression of Tendon Collagens and Tenocyte Markers in Long-Term Monolayer and High-Density Cultures of Rat Tenocytes

As a result of repeated movement, tendons are functionally open to traumas. According to this situation, tenocytes have already been used for tissue engineering therapies. It has been reported that long-term monolayer (ML) culture of tenocytes may lead to a phenotypic drift within passages. Depending on our previously published work, it is clearly demonstrated that high-density (HD) culture improves cell growth and differentiation of tenocytes. However, it is not yet established if HD favors the differentiated state during long-term culture. Therefore, we compared the differences in gene expression of tendon collagens and tendon markers of tenocytes from long-term ML and HD culture conditions by quantitative, real-time polymerase chain reaction (QRT-PCR) for over a period of 3 weeks. COLI, COLIII, COLV, Scx, and Tnmd were target genes as the major matrix constituents of tendons as well as being involved in matrix integrity and tenocyte phenotype. According to our results, tenocytes in HD culture synthesized less amounts of COLIII, COLV, and Tnmd, and dependent on the investigation time point, higher amounts of Scx. We consider that tenocytes produced in HD culture system may not provide sufficient efficiency during tissue engineering approaches. By the fact that most molecules showed significantly higher expression profiles in ML culture condition, it is suggested that culture and passage in ML should be taken into consideration for further tissue engineering approaches to maintain a phenotype with less amount of drift.

Effects of small abalone, Haliotis diversicolor, pedal mucus on bacterial growth, attachment, biofilm formation and community structure

Pedal mucus is important for locomotion, protection and signal transmission in the Gastropoda. It also provides nutrients and a habitat for microbes. In this study, we examined the effects of pedal mucus and the mucus trail of the small abalone, Haliotis diversicolor, on bacterial growth, attachment, biofilm formation and community structure. The results showed that pedal mucus enhanced the growth of bacteria, and that more opportunistic pathogens attached to a substrate covered with pedal mucus (for Vibrio alginolyticus, the cell density was up to 5 times higher than on a clean substrate). During microplate assay, when pedal mucus was added as the sole nutrient source, V. alginolyticus formed a biofilm, but few cells grew on the bottom of the wells if no mucus was added. Under flow-cell culture conditions, differently structured biofilms were formed in the clean and pedal mucus conditioned glass slides. The mucus trail induced a heterogeneous biofilm with cell clusters. Moreover, pedal mucus could support much more CFUs than the clean substrates. Using denatured gradient gel electrophoresis comparison of the bacterial community on a clean substrate and in the mucus trail, we found several specific species in the latter. At least one species, Klebsiella pneumoniae, is potentially harmful to the small abalone. In summary, the pedal mucus of Haliotis diversicolor enriched bacterial occurrence, including some pathogens, which deserves to be considered in high density culture conditions.

Ganglioside GM3 promotes cell migration by regulating MAPK and c-Fos/AP-1

Gangliosides have been proposed as modulators of transmembrane signaling. Recently, GM3, a glycosphingolipid containing monosaialic acids, is thought to be one of the key molecules of signal transduction in mammalian cells. In this study, we used mouse embryonic fibroblast cell lines (MEFs) established from sialyltransferase-I knockout mice (GM3 synthase KO mice) to evaluate the regulation of mitogenic signals by gangliosides. Cell proliferation assay revealed a higher growth potential of GM3 KO MEFs. Immunoblots showed upregulation of Ras/Raf/MEK/ERK pathway in GM3 KO MEFs, and these signals resulted in enhanced translocation of ERK into the nuclei. Further, both exogenous and endogenous add-back of GM3 decreased the activities of MAPK in GM3 KO MEFs. In addition, GM3 KO MEFs formed foci in high-density culture condition, and analyses of cell cycle modulators revealed the resistance of GM3 KO MEFs for entering cell cycle arrest. Finally, sustained expressions of c-Fos in GM3 KO MEFs were shown to correlate with DNA-binding activity between c-Fos and AP-1. These results demonstrate that the deletion of sialyltransferase-I changes the character of MEFs to a highly activated state of the MAPK pathway, indicating the critical role of GM3 as a regulator of membrane-transmitted signals.

In vitro neuronal differentiation of cultured human embryonic germ cells

Human embryonic germ (hEG) cells, which have been advanced as one of the most important sources of pluripotent stem cells [the other one being human embryonic stem cells], can be propagated in vitro indefinitely in the primitive undifferentiated state while being capable of developing into all three germ layer derivatives, hence have become anticipated developing novel strategies of tissue regeneration and transplantation in the treatment of degenerative diseases. In the experiments here, we derived hEG cells from cultured human primordial germ cells (PGCs) of 6- to 9-week-post-fertilization embryos. They satisfied the criteria previously used to define hEG cells, including the expression of markers characteristic of pluripotent cells-abundant alkaline phosphatase (AP) activity, stage specific embryonic antigen (SSEA)-1 +, SSEA-3 −, SSEA-4 +, TRA-1–60 +, TRA-1–81 +, Oct-4 +, and hTERT +, the retention of normal karyotypes, and possessing pluripotency by forming embryoid bodies (EBs) in vitro. Furthermore, these derived cells tended to neurally differentiate in vitro, especially under high-density culture conditions. We successfully isolated neural progenitor cells from differentiating hEG cultures and about 10% cells induced by 2 μM all- trans-retinoic acid (RA) or 0.1 mM dibutyryl cyclic AMP (dbcAMP)/1 mM forskolin to mature neurons expressing microtubule-associated protein 2ab (MAP2ab), synaptophysin, β-tubulin III, neuron-specific enolase (NSE), tyrosine hydroxylase (TH), but no glial fibrillary acid protein (GFAP) and choline acetyl transferase (ChAT). The data suggested that hEG cells may provide a potential source of cells for use in transplantation therapy for neurological degenerative diseases.

Behavior of embryonic rat cerebral cortical stem cells on the PVA and EVAL substrates

Cell behavior is determined by intrinsic programs and complex interactions among cells, medium components and substrates. Several previous reports have demonstrated the usefulness of extrinsic signals coming from soluble growth factors and cell–cell contact for regulating the proliferation and differentiation of neural stem cells. At present, the effects of substrate on neural stem cells are not known. In this study, the behavior of neural stem cells, isolated from embryonic rat cerebral cortex, was observed and compared on the polyvinyl alcohol (PVA) and poly (ethylene- co-vinyl alcohol) (EVAL) substrates in the presence of the mitogenic effect of basic fibroblast growth factor (bFGF) in the serum-free medium. It was found that PVA and EVAL exerted different influences on the fate of neural stem cells. The behavior of neural stem cells on the EVAL was independent of cell density at the single-cell level. Single neural stem cells seemed to remain dormant on the EVAL. Conversely, the development of cell clusters, termed neurospheres, was in a density-dependent manner on the EVAL. Neurospheres continuously proliferated under high-density culture condition, but differentiated into neurons and astrocytes under low-density culture condition. However, regardless of single cells or neurospheres, cultured cells could not survive on the PVA. Therefore, it is reasonable to assume that biomaterials may stimulate or inhibit the proliferation and differentiation of neural stem cells. These in vitro results are very encouraging since this information should be useful for the development of strategies for regulating the preservation, proliferation and differentiation of neural stem cells.

Developments in recirculating systems for Arctic char culture in North America

Arctic char (Salvelinus alpinus) tolerate high-density culture conditions, have an excellent fillet yield, are amenable to niche marketing, and are suitable for production within super-intensive recirculating systems. Much of the North American production of Arctic char has been within recirculating systems, which can provide more optimum water temperatures for fish growth and can also overcome limitations created by a lack of high-quality water resources or strict pollution discharge limits. This paper describes some of the developments that have been made in recirculating systems used to produce Arctic char and examines several North American facilities that have used recirculating systems to produce Arctic char. This description includes several state-of-the-art recirculating systems that are now being used to commercially produce Arctic char and another that has just been built and is about to come on-line. This paper also describes several areas where advances have been made in cold-water recirculating system design in order to improve the water quality that they maintain at high feed loadings and to increase the production capacity of these systems. Several critical process improvements include: increased hydraulic exchange rates through the culture tank, superior culture tank designs, better oxygen control strategies and ozonation, improved design of forced-ventilated cascade aeration columns, full flow drum filtration, and better pipe and sump cleanout designs. Several of the strengths and weaknesses of Arctic char production within land-based recirculating systems are also discussed.

The neuroprotective effects of estrogen in SK-N-SH neuroblastoma cell cultures

Estrogen has been considered to be a neuroprotectant and a neuromodulator in many neuronal cell lines and tissue preparations. The protective effects of estrogen may be mediated through classical estrogen receptors (ERs), or may be due to its anti-oxidant properties which are independent of receptors. The current studies show that 17β-estradiol (E2) is neuroprotective against β-amyloid protein 25–35 (Aβ)-, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-, high density culture condition-, and serum deprivation-induced neuronal death in SK-N-SH human neuroblastoma cells. SK-N-SH cells express ERβ, but not ERα, as detected by Western blot analysis. Among all the insults, MPTP, high density culture and serum deprivation induce apoptotic cell death in this cell system as detected by ELISA determination of mono/oligonucleosomes and DNA laddering, while Aβ induces necrotic cell death. The protective effects of E2 are abolished by the addition of tamoxifen and ICI 182,780 in the MPTP treated cells, but not in the other models, suggesting that the effect of E2 in the MPTP model is probably associated with activation of ERβ. The addition of ICI 182,780 shows a mitogenic effect in SK-N-SH cells in the presence of E2 in control culture or in the Aβ treated groups. Also, ICI 182,780 induced expression of ERα. Collectively, the current studies suggest that E2 is neuroprotective in apoptotic and necrotic death induced by multiple insults in SK-N-SH human neuroblastoma cells. Involvement of ER is insult type dependent. ICI 182,780 is able to influence the expression of ERs, probably through upregulation of ERα when ERβ is totally antagonized.

Rescue of Embryonic Epithelium Reveals That the Homozygous Deletion of the Retinoblastoma Gene Confers Growth Factor Independence and Immortality but Does Not Influence Epithelial Differentiation or Tissue Morphogenesis

The ability to rescue viable prostate precursor tissue from retinoblastoma-deficient (Rb-/-) fetal mice has allowed for the isolation and characterization of the first Rb-/- prostate epithelial cell line. This cell line, designated Rb-/-PrE, was utilized for experiments examining the consequences of Rb loss on an epithelial population. These findings demonstrated that Rb deletion has no discernible effect on prostatic histodifferentiation in Rb-/-PrE cultures. When Rb-/-PrE cells were recombined with embryonic rat urogenital mesenchyme and implanted into athymic male, nude mouse hosts, the recombinants developed into fully differentiated and morphologically normal prostate tissue. The Rb-/-PrE phenotype was characterized by serum independence in culture and immortality in vivo, when compared with wild type controls. Cell cycle analysis revealed elevated S phase DNA content accompanied by increased expression of cyclin E1 and proliferating cell nuclear antigen. Rb-/-PrE cultures also exhibited a diminished ability to growth arrest under high density culture conditions. We believe that the development of Rb-/- prostate tissue and cell lines has provided a unique experimental platform with which to investigate the consequences of Rb deletion in epithelial cells under various physiological conditions. Additionally, the development of this technology will allow similar studies in other tissues and cell populations rescued from Rb-/- fetuses.