Exposure to serotonin adversely affects oligodendrocyte development and myelination in vitro

Serotonin (5‐hydroxytryptamine, 5‐HT) has been implicated to play critical roles in early neural development. Recent reports have suggested that perinatal exposure to selective serotonin reuptake inhibitors (SSRIs) resulted in cortical network miswiring, abnormal social behavior, callosal myelin malformation, as well as oligodendrocyte (OL) pathology in rats. To gain further insight into the cellular and molecular mechanisms underlying SSRIs‐induced OL and myelin abnormalities, we investigated the effect of 5‐HT exposure on OL development, cell death, and myelination in cell culture models. First, we showed that 5‐HT receptor 1A and 2A subtypes were expressed in OL lineages, using immunocytochemistry, Western blot, as well as intracellular Ca²⁺ measurement. We then assessed the effect of serotonin exposure on the lineage development, expression of myelin proteins, cell death, and myelination, in purified OL and neuron‐OL myelination cultures. For pure OL cultures, our results showed that 5‐HT exposure led to disturbance of OL development, as indicated by aberrant process outgrowth and reduced myelin proteins expression. At higher doses, such exposure triggered a development‐dependent cell death, as immature OLs exhibited increasing susceptibility to 5‐HT treatment compared to OL progenitor cells (OPC). We showed further that 5‐HT‐induced immature OL death was mediated at least partially via 5‐HT2A receptor, since cell death could be mimicked by 5‐HT2A receptor agonist 1‐(2,5‐dimethoxy‐4‐iodophenyl)‐2‐aminopropane hydrochloride, (±)‐2,5‐dimethoxy‐4‐iodoamphetamine hydrochloride, but atten‐uated by pre‐treatment with 5‐HT2A receptor antagonist ritanserin. Utilizing a neuron‐OL myelination co‐culture model, our data showed that 5‐HT exposure significantly reduced the number of myelinated internodes. In contrast to cell injury observed in pure OL cultures, 5‐HT exposure did not lead to OL death or reduced OL density in neuron‐OL co‐cultures. However, abnormal patterns of contactin‐associated protein (Caspr) clustering were observed at the sites of Node of Ranvier, suggesting that 5‐HT exposure may affect other axon‐derived factors for myelination. In summary, this is the first study to demonstrate that manipulation of serotonin levels affects OL development and myelination, which may contribute to altered neural connectivity noted in SSRIs‐treated animals.

     

Transplantation of engineered bone tissue using a rotary three-dimensional culture system

Bone is a complex, highly structured, mechanically active, three-dimensional (3-D) tissue composed of cellular and matrix elements. We previously published a report on in situ collagen gelation using a rotary 3-D culture system (CG–RC system) for the construction of large tissue specimens. The objective of the current study was to evaluate the feasibility of bone tissue engineering using our CG–RC system. Osteoblasts from the calvaria of newborn Wistar rats were cultured in the CG–RC system for up to 3 wk. The engineered 3-D tissues were implanted into the backs of nude mice and calvarial round bone defects in Wistar rats. Cell metabolic activity, mineralization, and bone-related proteins were measured in vitro in the engineered 3-D tissues. Also, the in vivo histological features of the transplanted, engineered 3-D tissues were evaluated in the animal models. We found that metabolic activity increased in the engineered 3-D tissues during cultivation, and that sufficient mineralization occurred during the 3 wk in the CG–RC system in vitro. One mo posttransplantation, the transplants to nude mice remained mineralized and were well invaded by host vasculature. Of particular interest, 2 mo posttransplantation, the transplants into the calvarial bone defects of rats were replaced by new mature bone. Thus, this study shows that large 3-D osseous tissue could be produced in vitro and that the engineered 3-D tissue had in vivo osteoinductive potential when transplanted into ectopic locations and into bone defects. Therefore, this system should be a useful model for bone tissue engineering.     

Essential role of B-Raf in oligodendrocyte maturation and myelination during postnatal central nervous system development

Mutations in the extracellular signal-regulated kinase (ERK) pathway, particularly in the mitogen-activated protein kinase/ERK kinase (MEK) activator B-Raf, are associated with human tumorigenesis and genetic disorders. Hence, B-Raf is a prime target for molecule-based therapies, and understanding its essential biological functions is crucial for their success. B-Raf is expressed preferentially in cells of neuronal origin. Here, we show that in mice, conditional ablation of B-Raf in neuronal precursors leads to severe dysmyelination, defective oligodendrocyte differentiation, and reduced ERK activation in brain. Both B-Raf ablation and chemical inhibition of MEK impair oligodendrocyte differentiation in vitro. In glial cell cultures, we find B-Raf in a complex with MEK, Raf-1, and kinase suppressor of Ras. In B-Raf-deficient cells, more Raf-1 is recruited to MEK, yet MEK/ERK phosphorylation is impaired. These data define B-Raf as the rate-limiting MEK/ERK activator in oligodendrocyte differentiation and myelination and have implications for the design and use of Raf inhibitors.     

A root tissue culture system to study winter wheat-rhizobacteria interactions

Summary A tissue culture procedure was developed for growing winter wheat roots. This system was used to study bacterial-root interactions and root colonization by Pseudomonas cepacia strains R55 and R85, Azospirillum brasilense ATCC 29729 and Azotobacter chroococcum ATCC 9043. Axenic root tissue cultures were inoculated with bacteria and incubated at 25° C on a rotary shaker (150 rpm) for up to 3 weeks. At various intervals, root morphology and root hair development, bacterial colonization of root surfaces and nitrogenase activity were determined. As determined by plate counting techniques, bacterial attachment to the roots varied from 7.5 × 10⁴ to 3.2 × 10⁷ colony-forming units cm^–1. Scanning electron microscopy of inoculated roots revealed that some rhizobacteria (e.g., P. cepacia R85) significantly enhanced root hair development, and others (e.g., P. cepacia R55) specifically colonized root exudation sites. Nitrogenase activity of roots inoculated with either A. brasilense ATCC 29729 or A. chroococcum ATCC 9043 was stimulated if the inorganic nitrogen sources in the medium were replaced with 300 g glutamine ml^–1. We propose that the use of root tissue culture systems will facilitate studies on plant root-microbe interactions.Contribution no. R 626, Saskatchewan Institute of Pedology Offprint requests to: J. J. Germida     

A multi-station culture force monitor system to study cellular contractility

Cellular contraction contributes to the formation of scar tissue, which is characterized by an over-produced, disorganized collagen matrix. To study the contractility of cells in vitro and its potential contribution to scar tissue formation, we have developed a multi-station culture force monitor (CFM) system. This system consists of four vertical cantilever beams with semiconductor strain gages and a computerized data acquisition unit to monitor contractile forces of the cells in a collagen gel. Calibration showed that this system has a highly linear voltage-force relationship (R(2) > 0.99). Further, to demonstrate the applicability of this system, contractile forces of human skin fibroblasts in a collagen gel were measured. These fibroblasts were found to produce an average force of 0.2 nN/cell, which is consistent with the data in literature. The significant advantage of this CFM system is its ability to test multiple samples simultaneously. Therefore, the system can facilitate statistical design and analysis of experiments to study the effects of growth factors (e.g., TGF-betas) on cellular contraction and their potential role in scar tissue formation.     

Bi-zonal cartilaginous tissues engineered in a rotary cell culture system

In this study, we aimed at validating a rotary cell culture system (RCCS) bioreactor with medium recirculation and external oxygenation, for cartilage tissue engineering. Primary bovine and human culture-expanded chondrocytes were seeded into non-woven meshes of esterified hyaluronan (HYAFF-11), and the resulting constructs were cultured statically or in the RCCS, in the presence of insulin and TGFbeta3, for up to 4 weeks. Culture in the RCCS did not induce significant differences in the contents of glycosaminoglycans (GAG) and collagen deposited, but markedly affected their distribution. In contrast to statically grown tissues, engineered cartilage cultured in the RCCS had a bi-zonal structure, consisting of an outgrowing fibrous capsule deficient in GAG and rich in collagen, and an inner region more positively stained for GAG. Structurally, trends were similar using primary bovine or expanded human chondrocytes, although the human cells deposited inferior amounts of matrix. The use of the presented RCCS, in conjunction with the described medium composition, has the potential to generate bi-zonal tissues with features qualitatively resembling the native meniscus.     

Back to basics: using colour polymorphisms to study evolutionary processes

Here, I suggest that colour polymorphic study systems have been underutilized to answer general questions about evolutionary processes, such as morph frequency dynamics between generations and population divergence in morph frequencies. Colour polymorphisms can be used to study fundamental evolutionary processes like frequency‐dependent selection, gene flow, recombination and correlational selection for adaptive character combinations. However, many previous studies of colour polymorphism often suffer from weak connections to population genetic theory. I argue that too much focus has been directed towards noticeable visual traits (colour) at the expense of understanding the evolutionary processes shaping genetic variation and covariation associated with polymorphisms in general. There is thus no need for a specific evolutionary theory for colour polymorphisms beyond the general theory of the maintenance of polymorphisms in spatially or temporally variable environments or through positive or negative frequency‐dependent selection. I outline an integrative research programme incorporating these processes and suggest some fruitful avenues in future investigations of colour polymorphisms.     

A scale-dependent approach to study pollution control processes in wetland soils using three different techniques

The Elbe River, Germany, has received heavy metals and arsenic from the discharge of urban industrial, and agricultural effluent. During periods of inundation, these contaminants were transported with water into floodplain ecosystems, where they settled and accumulated predominantly in depressions and low-lying terraces. Markedly elevated arsenic concentration in soil solution during floods exceeded the inspection value of 10 μg L^?1 of the German soil protection ordinance. Highly variable hydrological conditions in floodplains can affect the dynamics of pollutants. The study of processes controlling the dynamics of pollutants is challenging because the results are required to answer both scientific and practical questions regarding protection of groundwater and plants, sustainable management of floodplains or explain the fate of environmentally harmful substances.Our experiments in small groundwater lysimeter and biogeochemical microcosms tended to yield similar results regarding the functional relationships among the investigated site parameters. But the results of the field experiments, carried out at a floodplain site of the middle course of the Elbe River, Germany, are often characterized by complex and varying factors. Whereas arsenic tended to be mobilized during flooding due to decreasing redox potential (E_H), chromium showed the opposite trend, with peak concentrations at the highest E_H values. Our approach at three different spatiotemporally scale levels, ranging from 23 days (microcosms) to two-and-a-half years (field soil hydrological facility) allows us to overcome process interferences observed in field studies.     

A lactococcal expression system for engineered nisins.

The nisin-producing Lactococcus lactis strain FI5876 has been modified and developed for use as an expression system for engineered nisin variants. Insertional inactivation of the resident nisA gene had a polar effect on downstream genes, including those involved in nisin immunity. However, subsequent chromosomal rearrangements in this region involving a newly discovered insertion element (IS905) generated a strain that was deficient in the nisA gene product but expressed those nisin determinants necessary for prenisin maturation, secretion, and immunity. Complementation of the lesion in the nisA gene by plasmid-encoded nisA genes containing site-specific mutations resulted in the exclusive production of altered nisins containing specific amino acid substitutions.     

A lactococcal expression system for engineered nisins.

The nisin-producing Lactococcus lactis strain FI5876 has been modified and developed for use as an expression system for engineered nisin variants. Insertional inactivation of the resident nisA gene had a polar effect on downstream genes, including those involved in nisin immunity. However, subsequent chromosomal rearrangements in this region involving a newly discovered insertion element (IS905) generated a strain that was deficient in the nisA gene product but expressed those nisin determinants necessary for prenisin maturation, secretion, and immunity. Complementation of the lesion in the nisA gene by plasmid-encoded nisA genes containing site-specific mutations resulted in the exclusive production of altered nisins containing specific amino acid substitutions.     

Multiple shoot culture of Dianthus caryophyllus using mist culture system

Summary A mist bioreactor system for the plant tissue cultures was developed. Using this system, the growth of Dianthus caryophyllus multiple shoots was directly measured. Tissue growth in mist bioreactor system was far better than that on agar medium and almost comparable to that in liquid medium. The mass increase (final dry weight/initial dry weight) in the mist culture was 2.85 while 3.28 in the liquid flask culture. Shoots were seriously vitrified in flask culture but these vitrifications could be considerably cured by using the mist culture system.     

A dense cell retention culture system using stirred ceramic membrane reactor

A novel reactor design incorporating porous ceramic tubes into a stirred jar fermentor was developed. The stirred ceramic membrane reactor has two ceramic tubular membrane units inside the vessel and maintains high filtration flux by alternating use for filtering and recovering from clogging. Each filter unit was linked for both extraction of culture broth and gas sparging. High permeability was maintained for long periods by applying the periodical control between filtering and air sparging during the stirred retention culture of Saccharomyces cerevisiae. The ceramic filter aeration system increased the k(L)a to about five times that of ordinary gas sparing. Using the automatic feeding and filtering system, cell mass concentration reached 207 g/L in a short time, while it was 64 g/L in a fed-batch culture. More than 99% of the growing cells were retained in the fermentor by the filtering culture. Both yield and productivity of cells were also increased by controlling the feeding of fresh medium and filtering the supernatant of the dense cells culture. (c) 1994 John Wiley & Sons, Inc.     

A two-stage bioreactor system for the production of recombinant proteins using a genetically engineered baculovirus/insect cell system

A two-stage bioreactor scheme was developed for the large-scale production of recombinant proteins using a genetically engineered baculovirus/insect cell system. The first bioreactor was employed for cell growth and the second for cell infection. Silkworm Bm5 cells were infected with a recombinant baculovirus, BmNPV/P5.cat, containing a bacterial chloramphenicol acetyltransferase (CAT) gene under the control of the polyhedrin gene promoter of Bombyx mori nuclear polyhedrosis virus (BmNPV). This recombinant baculovirus has been used as an expression vector for the production of recombinant CAT enzyme. A specific productivity of 82 to 90 mug CAT/(10(6) cells) was obtained using the BmNPV/Bm5 expression system, a yield similar to that achieved using the AcNPV/Sf expression system. Repeated infection of high-density cell cultures did not reduce the specific productivity of the CAT enzyme. Most importantly, the problems associated with the infection of high-density cell cultures were resolved by means of controlled infection conditions and appropriate replenishment of spent culture medium following infection. The glucose uptake rate by the cells following infection was 50% higher than that by the cells before infection. Not only did the infection of high-density cell cultures result in consistent yields of 250 mg/L of CAT enzyme, but also the two-stage bioreactor system was proven to be reliable for a long-term operation beyond 600 h. (c) 1993 John Wiley & Sons, Inc.     

Optimization of culture media to enhance the growth of tissue engineered cartilage

Tissue engineering is a promising option for cartilage repair. However, several hurdles still need to be overcome to develop functional tissue constructs suitable for implantation. One of the most common challenges is the general low capacity of chondrocytes to synthesize cartilage-specific extracellular matrix (ECM). While different approaches have been explored to improve the biosynthetic response of chondrocytes, several studies have demonstrated that the nutritional environment (e.g., glucose concentration and media volume) can have a profound effect on ECM synthesis. Thus, the purpose of this study was to optimize the formulation of cell culture media to upregulate the accumulation of cartilaginous ECM constituents (i.e., proteoglycans and collagen) by chondrocytes in 3D culture. Using response surface methodology, four different media factors (basal media, media volume, glucose, and glutamine) were first screened to determine optimal media formulations. Constructs were then cultured under candidate optimal media formulations for 4 weeks and analyzed for their biochemical and structural properties. Interestingly, the maximal accumulation of proteoglycans and collagen appeared to be elicited by different media formulations. Most notably, proteoglycan accumulation was favored by high volume, low glucose-containing DMEM/F12 (1:1) media whereas collagen accumulation was favored by high volume, high glucose-containing F12 media. While high glutamine-containing media elicited increased DNA content, glutamine concentration had no apparent effect on ECM accumulation. Therefore, optimizing the nutritional environment during chondrocyte culture appears to be a promising, straight-forward approach to improve cartilaginous tissue formation. Future work will investigate the combined effects of the nutritional environment and external stimuli.     

A cold-adapted protease engineered by experimental evolution system.

A new cold-adapted protease subtilisin BPN' mutant, termed m-51, was successfully isolated by use of an evolutionary program consisting of two-step in vitro random mutagenesis, which we developed for the screening of mutant subtilisins with increased activity at low temperature. The m-51 mutant showed 70% higher catalytic efficiency, expressed by the k(cat)/K(m) value, than the wild-type at 10 degrees C against N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide as a synthetic substrate. This cold-adaptation was achieved mainly by the increase in the k(cat) value in a temperature-dependent manner. Genetic analysis revealed that m-51 had three mutations, Ala-->Thr at position -31 (A-31T) in the prodomain, Ala-->Val at position 88 (A88V), and Ala-->Thr at position 98 (A98T). From kinetic parameters of the purified mutant enzymes, it was found that the A98T mutation led to 30% activity increase, which was enhanced up to 70% by the accompanying neutral mutation A88V. The A-31T mutation severely constrained the autoprocessing-mediated maturation of the pro-subtilisin in the Escherichia coli expression system, thus probably causing an activity-non-detectable mutation in the first step of mutagenesis. No distinct change was observed in the thermal stability of any mutant or in the substrate specificity for m-51. In the molecular models of the two single mutants (A88V and A98T), relatively large displacements of alpha carbon atoms were found around the mutation points. In the model of the double mutant (A88V/A98T), on the other hand, the structural changes around the mutation point counterbalanced each other, and thus no crucial displacements occurred. This mutual effect may be related to the enhanced activity of the double mutant.     

A simple wheat haploid and doubled haploid production system using anther culture

Summary Wheat (Triticum aestivum L.) haploids and doubled haploids have been used in breeding programs and genetic studies. Wheat haploids and doubled haploids via anther culture are usually produced by a multiple step culture procedure. We improved a wheat haploid and doubled haploid production system via anther culture in which plants are produced from microspore-derived embryos using one medium and one culture environment. In the improved protocol, tillers of donor plants were pretreated at 4°C for 1–2 wk before anthers were plated on a modified 85D12 basal medium with phenylacetic acid (PAA) and zeatin and cultured at 30°C with a 12-h daylength (43 μEs⁻¹m⁻²) in an incubator. Microspore-derived embryos developed in 2–3 wk and the plants were produced 3–4 wk after anther plating. In the improved system, as much as 53% of the anthers of Pavon 76 were responsive with multiple embryos. For plant regeneration, as many as 22 green and 25 albino plants were produced from 100 anthers. Sixty-five green plants were grown to maturity and 32 (49%) plants were fertile and produced seeds (indicating spontaneous chromosome doubling) while 33 plants did not produce seed. Of five Nebraska breeding lines tested using the protocol, NE96675 was very responsive and the other lines less so, indicating that the protocol is genotype-dependent.