Primary culture and characteristic morphologies of neurons from the cerebral ganglion of the mud crab, <Emphasis Type="Italic">Scylla paramamosain</Emphasis>

Crustacean neurons, obtained from the cerebral ganglion of the mud crab Scylla paramamosain, were successfully cultured in vitro. They maintained typical morphological characteristics and showed better outgrowth in modified Medium 199 (M199) medium than that in Liebowitz’s L-15 medium. Fetal bovine serum (FBS), muscle extracts, and hemolymph of the mud crab S. paramamosain were added as supplements. Only 20% FBS could promote neuron outgrowth, while muscle extracts and hemolymph of S. paramamosain did not improve neuron outgrowth. For cell dissociation, both collagenase type I and trypsin worked well as determined by initial cell viability and following cell outgrowth potential. More than six kinds of cells with different morphological characteristics were identified in the neuron outgrowth. They were “small cells”, “veilers”, “branchers”, “multipolar cells”, “super-large cell”, and “bipolar cells”. Among all of the cells, bipolar cells were identified for the first time in crustacean neurons culture and they could live longer than other cells. The neurons could grow for more than a week before retraction and eventual degradation.     

A new continuous cell line from larval ovaries of silkworm, <Emphasis Type="Italic">Bombyx mori</Emphasis>

A new continuous cell line from ovarian tissue of commercial variety “Kolar Gold” of silkworm, Bombyx mori, was established and designated as DZNU-Bm-12. The tissue was grown in MGM-448 insect cell culture medium supplemented with 10% fetal bovine serum (FBS) and 3% heat-inactivated B. mori hemolymph at 25 ± 1°C. The migration of partially attached small round refractive cells from the fragments of ovarioles began from the beginning of explantation. The cells multiplied partially attached in the primary culture initially, and some of them become freely suspended after 20 passages. The cells were adapted to MGM-448 and TNM-FH media each with 10% FBS and the population doubling time of cell line was about 36 and 24 hr, respectively. The chromosome number was near diploid at initial passages and slightly increased at 176th passage, but a few tetraploids and hexaploids were also observed. DNA profiles using simple sequence repeat loci established the differences between DZNU-Bm-12 and DZNU-Bm-1 and most widely used Bm-5 and BmN cell lines. The cell line was found susceptible to B. mori nucleopolyhedrovirus (BmNPV) with 85–90% of the cells harboring BmNPV and having an average of 3–17 OBs/infected cell. We suggest the usefulness of this cell line in BmNPV-based baculoviral expression system and also for studying in vitro virus replication.     

Microheological properties of different erythrocyte populations in persons with elevated arterial pressure and in physically active persons

Microrheological properties (aggregation and deformability) of erythrocytes separated by centrifugation at 30000×g density gradient into “young” and “old” (the upper and lower fractions, respectively) were studied. The erythrocytes were taken from physically active persons (PAP) and from those with elevated arterial pressure (EAP). A significant difference in microrheological properties of the “young” and “old” erythrocytes was found. The aggregation degree of “old” cells was nearly twice that of “young” cells. The deformability of the erythrocyte subpopulations was significantly different, though the difference was not so pronounced as in the case of aggregation. The aggregation of “young” and “old” erythrocytes in the PAP group was the least (28% lower than in the control). Note, that “old” erythrocytes in the PAP group had better microrheological properties than in the other groups. All erythrocyte populations in the EAP group were characterized by higher aggregation, decreased deformability, and decreased capacity for oxygen transport.     

An integrated strategy for the process development of a recombinant antibody-cytokine fusion protein expressed in BHK cells

Recombinant fusion proteins offer important new therapeutic approaches for the future. This report describes the use of three different genetic strategies (i.e. “mono-”, “bi-” and “tri-cistronic” vectors) to achieve stable secretion from BHK cells of a glycosylated antibody-cytokine fusion protein designed for use in anti-tumour therapy. It describes selection of a robust and effective production cell line based on stability of secretion of the product, quality of mRNA and protein products and performance in in vitro bioassays for potency. The data obtained at this stage were utilised in the selection of a suitable candidate production cell line. The relative productivity and general performance of the cells in stirred tank and fixed bed culture systems indicated that a variety of cell culture technologies provided robust tools for production of a highly selected cell clone. Consistency of the product glycosylation was determined by analysis of released oligosaccharides using matrix-assisted laser desorption ionisation – time of flight mass spectrometry and high-performance anion exchange chromatography. These investigations showed consistent expression of three glycoforms of the fusion protein which varied in their relative proportions in different culture systems and at different time points in a fixed bed reactor with continuous perfusion. In conclusion, this study dealt with a range of important scientific and technical issues which are essential for regulatory approval and commercial success of a recombinant protein and elucidates some useful markers for process development for similar recombinant biologicals. Received: 25 March 1999 / Accepted: 23 April 1999     

Characterization of some fish and shrimp spoiling bacteria

The classification of some important groups of bacteria involved in fish and shrimp spoilage was studied. Trimethylamine is produced byPseudomonas putrefaciens, a “non-defined” group resemblingPs. putrefaciens, Photobacterium spp. and someMoraxella-like bacteria. Hypoxanthine is produced by the same groups of bacteria except the last named and also by the “typical shrimp spoilers” (presumptiveAlteromonas). Strong off-odours are produced on fresh fish byPs. putrefaciens, dextroseoxidativePseudomonas spp. (Groups I and 11 according to Shewan, Hobbs and Hodgkiss, 1960), the above mentioned “non-defined” group and by only some of the “typical shrimp spoilers”, whereasMoraxella-like bacteria andPhotobacterium spp. failed to produce strong odours. Strong off-odours are produced on boiled shrimp by the “typical shrimp spoilers” (presumptiveAltermonas),Ps. putrefaciens, the dextrose-oxidativePseudomonas spp. and the “non-defined” group;Moraxella-like bacteria produced less offensive odours or none, nor didPhotobacterium.     

Detection and identification of the atypical bovine pestiviruses in commercial foetal bovine serum batches

The recently emerging atypical bovine pestiviruses have been detected in commercial foetal bovine serum (FBS) of mainly South American origin so far. It is unclear how widely the viruses are presented in commercial FBS of different geographic origins. To further investigate the possible pestivirus contamination of commercially available FBS batches, 33 batches of FBS were obtained from ten suppliers and analysed in this study for the presence of both the recognised and the atypical bovine pestiviruses. All 33 batches of FBS were positive by real-time RT-PCR assays for at least one species of bovine pestiviruses. According to the certificate of analysis that the suppliers claimed for each batch of FBS, BVDV-1 was detected in all 11 countries and BVDV-2 was detected exclusively in the America Continent. The atypical pestiviruses were detected in 13 batches claimed to originate from five countries. Analysis of partial 5'UTR sequences showed a high similarity among these atypical bovine pestiviruses. This study has demonstrated, for the first time that commercial FBS batches of different geographic origins are contaminated not only with the recognised species BVDV-1 and BVDV-2, but also with the emerging atypical bovine pestiviruses.     

Modeling of the actomyosin ATPase activity

In the rapid “quench” kientics of myosin, the “initial phosphate burst” is the excess inorganic phosphate that is produced during the early time-course of ATP hydrolysis by myosin subfragment-1 (S-1) or HMM. In general, the existence of a Pi burst implies a rapid (i.e., generally an order of magnitude faster than the steady-state hydrolysis rate) lysis of the phospho-anhydride bond within the ATP molecule, followed by one or more slower steps that are rate limiting for the process. Thus, the presence of a Pi burst can provide an important clue to the mechanism of the reaction. However, in the case of actomyosin, this clue as long been the subject of controversy and misunderstanding. To measure the (initial) Pi burst, myosin S-1 (or HMM) is rapidly mixed with ATP and then the mixture is acid quenched after a specific time period. The medium produced contains free Pi generated from hydrolysis of the ATP. The quantitative measure of the phosphate generated in this way has always been significantly greater than that expected by steady-state “release” of Pi alone, and it is that very difference between this measured Pi after the quench and that amount of Pi expected to be released by steady-state considerations in that same time period that has been referred to as the “initial Pi burst”. Recent investigations of the kinetics of Pi release have used an entirely new method that directly measures the release of Pi from the enzyme-product complex. These studies have made reference to the properties of the “initial Pi burst” in the presence of actin, as well as to a new kinetic entity: the “burst of Pi release”, and have been often vague concerning the true nature of the initial Pi burst, as well as the properties of Pi release as predicted by the current models of the actin activation of the myosin ATPase activity. The purpose of the current article is to correct this oversight, to discuss the “burst” in some detail, and to display the kinetics predicted by the current models for the actin activation of myosin. Furthermore, predictions for the kinetics of the new “burst of Pi release” are discussed in terms of its ability to discriminate between the two current competing models for actin activation of the myosin ATPase activity.     

Cultures of human tracheal gland cells of mucous or serous phenotype

There are two main epithelial cell types in the secretory tubules of mammalian glands: serous and mucous. The former is believed to secrete predominantly water and antimicrobials, the latter mucins. Primary cultures of human airway gland epithelium have been available for almost 20 yr, but they are poorly differentiated and lack clear features of either serous or mucous cells. In this study, by varying growth supports and media, we have produced cultures from human airway glands that in terms of their ultrastructure and secretory products resemble either mucous or serous cells. Of four types of porous-bottomed insert tested, polycarbonate filters (Transwells) most strongly promoted the mucous phenotype. Coupled with the addition of epidermal growth factor (EGF), this growth support produced “mucous” cells that contained the large electron-lucent granules characteristic of native mucous cells, but lacked the small electron-dense granules characteristic of serous cells. Furthermore, they showed high levels of mucin secretion and low levels of release of lactoferrin and lysozyme (markers of native serous cells). By contrast, growth on polyethylene terephthalate filters (Cyclopore) in medium lacking EGF produced “serous” cells in which small electron-dense granules replaced the electron-lucent ones, and the cells had high levels of lactoferrin and lysozyme but low levels of mucins. Measurements of transepithelial resistance and short-circuit current showed that both “serous” and “mucous” cell cultures possessed tight junctions, had become polarized, and were actively secreting Cl.     

Assessment of the Neva Estuary ecosystem state on the basis of structural characteristics of benthic animal communities in 1994–2005

In the years 1994–2005, the values of the integrated index IP’ at some stations of the Neva Bay changed from 38.1 to 81.9%, water quality changed from class 3 to class 5, and the states of some areas of the ecosystem that were evaluated as “tense” went to “catastrophic.” The integrated mean assessment of water quality according to the IP’ index over the entire Neva Bay throughout 12 years (1994–2005) remained relatively stable, waters were assessed as “polluted” (fourth class), and the state of the ecosystem was considered “critical.” The state of the eastern part of the Gulf of Finland in 1994–2005 was less favorable. The species diversity of zoobenthos in the resort zone of the eastern part of the Gulf of Finland is considerably lower than in the Neva Bay. Waters of the resort zone of the eastern part of the Gulf of Finland in 1994–2005 were assessed as one class lower than in the Neva Bay, i.e., as “polluted-dirty” (fourth-fifth class), and the state of the ecosystem was assessed as being in a “crisis.” In the resort zone, there was a decline in species diversity and abundance and biomass of benthic animals; i.e., all characteristics of the degradation of benthic animal communities were observed.     

Subpopulation of nestin positive glial precursor cells occur in primary adult human brain cultures

Glial fibrillary acidic protein (GFAP) is an intermediate filament protein considered to be the best astroglial marker. However, the predominant cell population in adult human brain tissue cultures does not express GFAP; these cells have been termed “glia-like” cells. The basic question about histological origin of adult human brain cultures remains unanswered. Some authors showed that “glia-like” cells in adult human brain cultures might be of non-glial origin. We examined primary explant tissue cultures derived from 70 adult human brain biopsies. Within first 5–10 days approximately 5–10% of the small explants became attached. Outgrowing cells were mostly flat cells. These cells formed confluent layer over 3–6 weeks in culture. At confluence the cultures contained 2–5% of microglial cells, 0.1% GFAP-positive astrocytes, less than 0.01% oligodendrocytes and 95–98% GFAP-negative “glia-like” cells. This population of flat “glia-like” cells was positively stained for vimentin, fibronectin, and 20–30% of these cells stained for nestin. Our findings revealed that 1 mM dibutyryl-cAMP addition, in serum free conditions, induced a reversible stellation in 5-10% of the flat “glia-like” cells but did not induce the expression of GFAP or nestin in morphologically changed stellate cells. These results demonstrate that “glia-like” cells in primary adult human brain cultures constitute heterogeneous cell populations albeit with similar morphological features. Two distinct subpopulations have been shown: (i) the one immunostained for nestin; and (ii) the other reactive for dibutyryl-cAMP treatment.     

Ultrastructure of the carapace margin in the Ostracoda (Arthropoda: Crustacea)

In podocopid ostracods, the “hingement”, with teeth/sockets or crenulations, is developed along the attached dorsal margin of the carapace. Dual calcified lamellae, called “duplicatures” are also developed along the free margin. However, the terminology of these marginal areas is often confused by many ostracodologists because of their differing interpretations and viewpoints. In this study, the marginal areas of the carapace were observed in detail, using the transmission (TEM) and scanning (SEM) electron microscopes. Consequently, the terminology of the marginal areas has been revised. The homology between attached and free margins and the structural differences of duplicatures, for some taxa are also discussed.     

Assessment of Gene Flow from Genetically Modified Anthracnose-Resistant Chili Pepper (Capsicum annuum L.) to a Conventional Crop

We conducted a 2-year field assessment of the gene flow from genetically modified (GM) chili pepper (Capsicum annuum L.), containing the PepEST (pepper esterase) gene, to a non-GM control line “WT512” and two commercial hybrid cultivars, “Manidda” and “Cheongpung Myeongwol (CM).” After seeds were collected from the pollen-recipient non-GM plants, hybrids between them and the GM peppers were screened by a hygromycin assay. PCR with the targeting hpt gene was performed to confirm the presence of transgenes in hygromycin-resistant seedlings. Out of 7,071 “WT512” seeds and 6,854 “Manidda” seeds collected in 2006, eight and 12 hybrids, respectively, were detected. In 2007, 33 hybrids from 3,456 “WT512” seeds and 50 hybrids from 3,457 “CM” seeds were found. The highest frequency of gene flow, 6.19%, was observed in that 2007 trial. These results suggest that a limited isolation distance would be sufficient to prevent gene flow from GM to conventionally bred chili peppers.     

Abscisic acid metabolism and lipid accumulation of a cell suspension culture of <Emphasis Type="Italic">Lesquerella fendleri</Emphasis>

Lesquerella fendleri (commonly known as “Fendler’s bladderpod” or “yellowtop”) is a member of the Brassicaceae and is an important seed oil-producing plant. The lipid profile of L. fendleri seed indicates potential for producing a high quality replacement for castor oil. In this work, characterization of the lipid content of a suspension cell culture, derived from seedlings of L. fendleri, is provided. Under the described suspension cell culture conditions, 16:0, 18:1Δ9, 18:2 Δ9, Δ12 and 18:3 Δ9, Δ12, Δ15 fatty acids were found to accumulate in the cells, while 16:0, 26:0 and 28:0 fatty acids were predominant in the culture medium. Subsequently, the effect of application of abscisic acid (ABA), which modulates lipid accumulation, was assessed. Exogenously applied ABA was taken up by the cells and metabolized via the conjugation pathway, resulting in the accumulation of ABA-glucose ester. Preliminary tests demonstrate the cell line is responsive to exogenous ABA, resulting in increased cellular lipid content and increased accumulation of lipids in the culture medium. This novel L. fendleri suspension culture presents a valuable model system for efficient characterization of mechanisms associated with ABA-induced accumulation of lipids.     

Dynamics of differentiation in human epidermoid squamous carcinoma cells (A431) with continuous,long-term γ-IFN treatment

Summary We investigated the long-term effects of continuous gamma interferon (γ-IFN) treatment on A431, a human squamous carcinoma cell line. Cells were grown in an in vitro culture system, which over time produces cohesive cell masses (“tumoroids”) exhibiting three-dimensional, histotypically differentiated structures, e.g., keratin “pearls,” intercellular bridges (desmosomes), elongated flattened cells (squames) and stratification. The effects of γ-IFN on cell growth, morphology and stage of differentiation were assessed at different treatment times by light and electron microscopy and by immunohistochemical staining using antibodies to keratins 1 and 14 and to filaggrin, markers of specific stages of keratinocyte differentiation. Our results show that A431 cells have the capacity for spontaneous differentiation, that this capacity is significantly enhanced and accelerated by γ-IFN treatment leading to terminal differentiation and extensive cell death by 2 wk. Despite continuous exposure to IFN, a small number of viable, undifferentiated cells remain. Their proliferation, evident by 3 wk, reconstitutes the tumoroid which once again contains the full range of differentiating cell types.     

FGF2 and dexamethasone increase the production of hyaluronan in two-dimensional culture of elastic cartilage-derived cells: in vitro analyses and in vivo cartilage formation

Elastic cartilage-derived cells cultured two-dimensionally with FGF2 and corticosteroid produce gel-type masses that become mature cartilage when injected into a subcutaneous pocket. This unique method has previously been clinically applied for treatments of nasal augmentation. However, the components of the gel-type mass and the mechanism of its synthesis remain unknown. Here, we have investigated the components of the gel-type mass produced by elastic cartilage-derived cells, and whether this gel-type mass can be produced by using other cell sources or other media. Human elastic cartilage-derived cells from auricular cartilage, hyaline cartilage-derived cells from articular cartilage, and mesenchymal stem cells from synovium were cultured in three media: “redifferentiation medium” containing FGF2 and dexamethasone; “chondrogenic medium” containing bone morphogenetic protein-2, transforming growth factor-β3, and dexamethasone specific for in vitro chondrogenesis of mesenchymal stem cells; control medium. The elastic cartilage-derived cells cultured in redifferentiation medium produced a gelatinous matrix positive for Alcian blue. During culture, the amount of chondroitin 4-sulfate, chondroitin 6-sulfate, and especially hyaluronan increased. However, the expression of RNAs for most chondrogenic genes did not increase. We also reproduced cartilage tissue formation by the injection of elastic cartilage-derived cells with the gelatinous mass into the subcutaneous space of the nude mouse. The synthesis of gelatinous matrix in vitro and the formation of cartilage tissue in vivo could be obtained only for the combination of elastic cartilage-derived cells with redifferentiation medium. This study was supported in part by grants from the “Japan Society for the Promotion of Science (19591752)” and “Center of Excellence Program for Frontier Research on Molecular Destruction and Reconstruction of Tooth and Bone in Tokyo Medical and Dental University” to Takeshi Muneta, and the “Japan Society for the Promotion of Science (18591657)” to Ichiro Sekiya.     

Basidiocarp development inCoprinus macrorhizus

The dikaryon #5026 (A2B2)+#5132 (A7B7) of the basidiomyceteCoprinus macrorhizus was treated with ultraviolet light (UV),N-methyl-N′-nitro-N-nitrosoguanidine (NG), and 5-bromouracil (BU) in order to induced developmental variations in fruiting. Among a total of 11,383 isolates, 742 were homokaryons. Fruiting test was made with the remaining 10,641 dikaryotic isolates, of which 1,594 showed abnormality in basidiocarp development. Both UV and NG were very efficient in inducing such variations, but BU was not. The induced variations were classified into seven basic types as follows: 1) “knotless”, in which hyphal knots, the first sign of fruiting, do not differentiate; 2) “primordiumless,” in which hyphal knots are formed, but no further development occurs; 3) “maturationles”, in which primordia are formed, but the maturation of primordia into adult fruit bodies does not occur; 4) “elongationless”, in which the elongation of stipe is blocked, and a basidiocarp with a very short stipe is produced; 5) “expansionless,” in which pilei do not expand normally; 6) “sporeless,” in which the formation of basidiospores is blocked and albinic pilei bearing none or only a small amount of spores are produced; and 7) “autolysisless,” in which the autolysis of pilei does not appear to occur. It has been noticed that the four steps of maturation, i.e. stipe elongation, pileus expansion, spore formation, and perhaps pileus autolysis, can proceed independently. Compound types of variations such as “elongation-expansionless,” “elongation-sporeless,” “expansion-sporeless,” and “elongation-expansion-sporeless” were also induced. UV treatment induced maturationless at the highest rate, while NG treatment sporeless. Contributions from the Division of Plant Morphology, Department of Biology, Faculty of Science, Okayama University. No. 117.     

In vitro separation of a rose chimera

“Fairmount 1 thorny” (“FM1 thorny”) (a Rosa multiflora Thunb ex. J. Murr.) and a thornless sport of “FM1 thorny” (“Fairmount 1” (“FM1”)) were established in vitro to investigate chimeral segregation under various levels of BA and to obtain a pure thornless rose. While the chimeral thornless sport was expected to segregate in vitro and yield both thorny and thornless plantlets, “FM1 thorny” was to yield only thorny plants. “FM1” segregated in vitro into its constituent genotypes and yielded thorny and thornless plantlets, suggesting that “FM1” is chimeral. “FM1 thorny” produced only thorny plants in vitro. These results indicate that the “FM1 thorny” clone was not chimeral (pure thorny) and that the thornless regenerates of “FM1” did not develop via somaclonal variation. There was a significant linear relationship between increasing BA concentration and the percentage of thorny plants. Among a population of 690 tissue culture derived plants from all the BA experiments, 6 plants were classified as pure thornless plants 1 year later.     

Impact of Willow Short Rotation Coppice on Water Quality

Short rotation coppice (SRC) with willow has been grown in Sweden from the late 1980s to produce biomass for energy on agricultural land. This study evaluated the effects of SRC on water quality by determining differences in leaching of nitrogen and phosphorus to groundwater of a number of commercial “old” SRC willow stands in Sweden compared to adjacent arable fields grown with “ordinary” crops. The study was conducted in 16 locations under three vegetation seasons. NO₃–N leaching from willow SRC fields was significantly lower than that from reference fields with cereals. The opposite was observed for PO₄–P; concentrations in the groundwater of SRC were higher compared to reference fields. Sewage sludge applications were not responsible for the elevated PO₄–P leaching under SRC compared to reference crops.     

Study on high-temperature-resistant and high-yield <Emphasis Type="Italic">Laminaria</Emphasis> variety “Rongfu”

Hybridization of gametophytes, continuous self-crossing and targeted selection were utilized to breed a new Laminaria variety. After five-generation selection breeding, the new variety “Rongfu” was obtained. Its male parent “Yuanza No.10” was the high-yield cultivation variety, and its female parent was variety “Fujian” which could tolerate relatively high seawater temperature. “Yuanza No.10” and “Fujian” were different but complement in their morphological characteristics and biological habits. Variety “Rongfu” was bred through their hybridization which exhibited high-yield potential and high seawater temperature tolerance. The results of traits evaluation in consecutive years showed that “Rongfu” attained higher yields by 24–27% compared to the control (widely used commercial variety) and also contained considerable amounts of iodine, mannitol, and algin. When seawater temperature was 18–21°C, the blade growth of “Rongfu” was maintained and tissue loss by abrasion was significantly lower than the control. Since the adoption of variety “Rongfu” in 2001, its cultivation areas have been extended to Shandong, Fujian and Guangdong province and have reached 14,133 ha currently, i.e., almost one-tenth of the total cultivation areas of Laminaria in China. The results of Random Amplified Polymorphic DNA analysis showed that the relationship between “Rongfu” and other cultivation varieties in China was very close.     

Altered free calcium transients in pig kidney cells (LLC-PK1) cultured with penicillin/streptomycin

Summary The effect of a conventional antibiotic (penicillin/streptomycin) mixture on the widely used kidney epithelial cell line, LLC-PK₁, was investigated by measuring growth and intracellular free calcium. Free calcium concentration was the same in cells cultured for 3 to 7 wk with (“plus”) and without (“minus”) antibiotics both at rest and when challenged with high (14 mM) external calcium. When exposed to vasopressin, minus cells exhibited significantly smaller calcium transients than plus cells. A similar difference existed for transients elicited by a calcium ionophore, 4-br-A23187. After longer periods of culture (>20 wk), minus cells grew slower than plus cells but on reaching confluence (minus cells took 1 day longer) the morphologies and viabilities were indistinguishable. The finding that culture with penicillin/streptomycin reversibly modified some properties of LLC-PK₁ cells, at least partly through altered calcium homeostasis, is of importance for workers using this cell model to study drug effects and raises the general possibility of similar effects on other cultured cells.