Retention of the differentiated state by larvalXenopus liver cells in primary culture

Summary Electron microscopic analysis of primary cultures derived from larvalXenopus liver has shown that these cells, although they form only two-dimensional aggregates, retain and presumably also develop structural characteristics typical of liver parenchyma cells, such as bile canaliculi with microvilli and epithelial junctional complexes. As judged from structural criteria, primary cultures contain 80–90% hepatocytes. In contrast to the intact tissue, primary cultures showed excessive development of microfilaments, however.Incorporation of labeled amino acids has revealed further that the capacity for protein synthesis is maintained in culture and that synthesis of liverspecific protein albumin is maintained in vitro, even in liver cultures derived from thyrostatic tadpoles. This latter result suggests that initiation of albumin synthesis in the larval liver is probably not dependent upon thyroid hormones but rather reflects the protodifferentiated state of this tissue.     

Isolation of plasma membrane complexes fromXenopus oocytes

Summary A method for the isolation of plasma membrane fractions fromXenopus oocytes has been developed, and the membranes have been characterized biochemically and morphologically. Plasma membrane complexes prepared by this procedure consisted of large sheets of the membrane, with associated vitelline envelope (a nonmembranous meshwork of fibers) and cortical (secretory) granules still attached. The morphology of cell surface microvilli and coated pits was well preserved. Cortical granules were removed by gentle homogenization in a low ionic strength medium, and integral and peripheral membrane proteins were then separated from vitelline envelopes by detergent extraction and phase separation in Triton-X-114. Biochemical characterization of the plasma membrane fractions indicated substantial levels of 5-nucleotidase and alkaline phosphodiesterase activity associated with the oocyte cell surface, with 44–66% recovery of these markers in the final membrane preparations. Lectin blotting and lectin affinity chromatography with Concanavalin A and wheat germ agglutinin were used to characterize the major glycoprotein species associated with the plasma membrane complexes. Plasma membrane fractions prepared by this procedure should be very useful in both biochemical and morphological studies of membrane protein sorting in theXenopus oocyte system.     

Radiation stimulated increase of plating efficiency of free plant cells

Summary Radiation induced stimulation of plating efficiency of free plant cells was observed following irradiation with X-rays (1.25 Gy, dose rate 3.1 Gy.nin^–1) and fission neutrons (1.5 Gy, dose rate 0.05 Gy.nin^–1). The dose range where the radiation stimulation effect is manifest is inversely correlated with the applied dose rate.The results are discussed in view of the radiation induced stimulation as it is applied in agricultural practice.     

Characterization of a new cell line,XL2, obtained fromXenopus laevis and determination of optimal culture conditions

Summary A new amphibian permanent cell line is described. It is called XL2 and was initiated from Stage 35 tadpoles ofXenopus laevis. The cell line has an epithelioid morphology and most cells can be classified into two populations with respective chromosome modal numbers 36 and 74. Contact inhibition is low. Its growth is vigorous in L15 or MEM medium supplemented with fetal bovine serum. The mean doubling time is 39 hr and the saturation density is 700,000 cells/cm² at 25°C. The absolute plating efficiency is about 70%. Cell line XL2 is unable to grow in L15 medium containing a macromolecular fraction of fetal bovien serum. Growth is restored if the latter medium is supplemented with 10 μg/ml of hypoxanthine. Optimal conditions for the dye exclusion test, for harvesting the cells, and for cloning in petri dishes are described. This work was supported by Grant 3,4514,79 of the Fonds de la Recherche Scientifique Médicale (Belgium) and by a grant of the Fonds de Développment Scientifique of the Catholic University of Louvain. The authors are indebted to Mrs. M. S. Denis, Mr. F. Desneux, and Ms. J. Janssens for competent technical assistance. Smith Kline-RIT, Genval (Belgium), is acknowledged for the generous gift of antibiotics and for performing the cultures for mycoplasma detection.     

Estrogen-dependent DNA synthesis in cultures ofxenopus liver parenchymal cells

In Vitro Cellular &; Developmental Biology - Plant - We have recently shown that extensive proliferation of liver parenchymal cells takes place in adult maleXenopus frogs in response to...     

Factors affectingin vitro establishment and shoot proliferation ofRosa hybrida L. andRosa Chinensis minima

Summary The effects of nodal explants collected at different plastochrones, use of various benzyladenine (BA) concentrations, sources of carbohydrates, and phases of the culture medium on shoot establishment and proliferation ofRosa hybrida L. andR. chinensis minima were evaluated. Higher numbers of shoots per explant were obtained fromR. hybrida cv. Carefree Beauty explants proximal to the apical meristem than those from distal nodes. However, proliferating shoots derived from plastochrones proximal to the apical meristem had a lower number of leaves/explant and were shorter than those derived from other distal plastochrones. Although shoot proliferation increased with higher BA concentration in the medium, a concentration of 4.4 μM BA was found optimum for axillary bud-break and shoot development forR. hybrida cvs. Adelaide Hoodless and Cuthert Grant. A higher shoot proliferation rate was observed forR. hybrida cv. Carefree Beauty explants grown on a medium containing 55.5 mM fructose than 58.4 mM sucrose. However, no differences were observed forR. hybrida cv. Cuthert Grant grown on a medium containing either fructose or sucrose. The mean number of shoots/explant was higher forR. chinensis minima cv. Red Sunblaze explants grown on a liquid (4.5) than on a solid medium (1.7) for the first reculture; while no significant differences between the two phases of the medium were observed for the second reculture. However, a higher mean number of shoots/explant was observed on solid-phase (4.0) than liquid-phase medium (3.4) for the third reculture. A higher mean number of leaves/shoot was obtained on a solidified medium rather than liquid medium in the first two consecutive recultures, while no differences were observed for the third reculture. Although a significant effect of BA concentration on mean number of shoots/explant was observed for Red Sunblaze nodal explants, the influence of BA concentration decreased in the two consecutive cultures for both phases of the medium. Hyperhydricity was observed on Red Sunblaze shoots grown on the liquid-phase medium.     

Streptomycin toxicity in primary cultures of flounder renal proximal tubule cells

Summary The aminoglycoside antibiotic streptomycin is a known nephrotoxin in vivo and a common component of cell culture media. The effects of streptomycin (100 μg/ml) on transepithelial electrical properties, glucose transport, glycolytic metabolism, and morphology were examined in primary proximal tubule cell cultures from winter flounder (Pseudopleuronectes americanus) kidney. Streptomycin treatment on either Days 2 to 12 or Days 8 to 13 abolished the transepithelial potential difference and short-circuit current across the monolayer but had no effect on transepithelial resistance in confluent 12 to 13-dcultures, suggesting the loss of active transepithelial transport. Consistent with these findings, mucosal-to-serosal glucose fluxes were greatly reduced in streptomycin-treated cultures and insensitive to the transport inhibitor phlorizin, indicating the absence of the apical Na-dependent glucose transport system associated with net glucose reabsorption. In addition to transport processes, antibiotic treatment also interfered with cellular energy metabolism as judged by the rapid reduction in glycolytic lactate production observed in the presence of streptomycin. Scanning and transmission electron microscopy revealed that streptomycin-treated culture were composed of cuboidal-to-columnar shaped cells which maintained intact tight junctions similar to control cultures. However, apical microvilli, the presumed sites of mucosal transport systems, were severely reduced in number in streptomycin-treated cultures. We concluded that streptomycin, at a dose commonly used in cell culture, inhibited the expression of differentiated function by flounder proximal tubule cell cultures. These cell cultures may provide a suitable model system for examination of the mechanisms of aminoglycoside nephrotoxicity. This investigation was supported by the University of Connecticut Research Foundation and by grant PCM-8003452 from the National Science Foundation, Washington, DC.     

Cell proliferation in ectodermal explants from Xenopus embryos

Summary Dissociated prospective ectoderm cells from Xenopus laevis embryos divide autonomously up to the 17th division cycle of the embryo. To examine the requirements for the further proliferation of these cells, the continuation of cell division in compact ectodermal explants beyond the 17th division cycle has been studied. Such explants develop into aggregates of epidermal cells, as can be shown immunohistochemically with an anti-serum against Xenopus epidermal cytokeratin. Cell division in these explants is comparable to the in vivo proliferation rate at least during the first 24 h of cultivation, that is, well beyond the 17th division cycle. Thus, epidermal cells are provided with all the factors necessary for continued proliferation, but these can be effective only when the cells form tight aggregates. The long-term changes in cell number are complex. Mitotic figures are present until the explants disintegrate after 3–4 days. However, the total cell number per explant does not increase during later development. The production of cells by mitotic divisions is likely to be countered by the loss of cells due to cell death, which is indicated by the presence of pyknotic nuclei.     

Characterization of mussel gill cells in vivo and in vitro

Mussel gill cells are attractive models in ecotoxicological studies because gills are the first uptake site for many toxicants in the aquatic environment; gill cells are thus often affected by exposure to pollutants. Our aim was to characterize mussel gill cells in vivo and in vitro by using morphological, histochemical and functional end-points. In paraffin sections stained with haematoxylin–eosin, three zones were distinguished in the long central gill filaments: frontal, intermediate and abfrontal. Various types of ciliated cells were present in the frontal zone, and both ciliated and non-ciliated cells were found in the abfrontal zone. The intermediate zone was comprised of flattened endothelial cells. Lipofuscin granules occurred in the three zones in variable amounts, depending on the specimen. Haemocytes were found in the haemolymph sinus of gill filaments. Mucocytes were identified in both frontal and abfrontal zones by means of periodic acid Schiff-alcian blue (PAS-AB) staining. In cryostat sections, succinate dehydrogenase (SDH) activity was mainly found in ciliated cells, whereas neutral lipids and acid-phosphatase-reactive lysosomes were present in all portions of the gill filament, mostly being related to lipofuscin granules. In mussels exposed to 5-bromo-2-deoxyuridine in vivo, proliferating cells were scattered throughout the gill filament. Gill cells (typically 2×10⁷ cells/ml per mussel; 95% viability) were isolated by dissociation with dispase. Gill cell suspensions were heterogeneous: 58% were ciliated epithelial cells (positive for SDH), 42% were non-ciliated cells (including epithelial cells and haemocytes), 2.3% were mucocytes (positive for PAS-AB) and 4.25% were haemocytes (able to phagocytose neutral red-stained zymosan). Gill cell cultures were maintained up to 18 days without changing the culture medium, viability decreasing below 50% at day 18. Primary cultures of mussel gill cells might therefore be useful models for the in vitro assessment of xenobiotic impacts on coastal and estuarine ecosystems.This work was funded by the Spanish Ministry of Science and Technology (project AMB99-0324), by the Basque Government through the Cooperation Fund Aquitaine/Euskadi 2001, by the University of the Basque Country through a grant to Consolidated Research Groups and by the European Commission (BEEP project, contract no. EVK3-CT2000-00025). Amagoia Gómez-Mendikute is the recipient of a predoctoral fellowship from the Spanish Ministry of Education and Culture.     

The effects of BmNPV on biochemical changes in primary cultures of Bombyx mori embryonic tissue

The effect of Bombyx mori nuclear polyhedrosis virus (BmNPV) on biochemical changes of TC-100 medium containing 10% fetal bovine serum (FBS) in embryonic primary cultures of silkworm was investigated. The primary cultures that reached 60% confluence were infected by 0.5, 1, and 2-ml viral inoculums (diluted with TC-100 medium representing multiplicity of infection (MOI) of 0.25, 0.5, and 1). Glucose, uric acid, urea, total protein, cholesterol, and alkaline phosphatase were measured in the medium of BmNPV-infected primary cultures. All biochemical compounds showed significant changes. Glucose decreased considerably by about 55 mg/ml, while different concentrations of the virus inoculums did not demonstrate significant differences among them. Total protein had only increased in 2 ml concentration and there were no changes in other concentrations. Uric acid as a by-product accumulated dramatically in all concentrations, while the amount of urea reduced in all treatments and this reduction was more evident in lower concentrations. Cholesterol consumption was high in cultures postinfection, while alkaline phosphatase (ALP) activity decreased in infected cells.     

Rosmarinic acid production in Coleus cell cultures

Cell suspension cultures of Coleus blumei Benth. have been found to accumulate 8–11% of their dry weight as rosmarinic acid (-O-caffeoyl-3,4-dihydroxyphenyl-lactic acid). Actively-growing tissue converts >20% of exogenously supplied phenylalanine and tyrosine to the caffeoyl ester and this high rate of synthesis coincides with an increase in phenylalanine ammonia-lyase specific activity. Administration to the cultures of known phenylpropanoid precursors of rosmarinic acid failed to enhance the latter's production and in some cases inhibited it.Abbreviations RA rosmarinic acid (-O-caffeoyl-3,4-dihydroxyphenyllactic acid - DOPA dihydroxyphenylalanine - PAL phenylalanine ammonialyase - DOPL dihydroxyphenyl-lactic acid     

Establishment of primary cell cultures from fish calcified tissues

Fishes have been recently recognized as a suitable model organism to study vertebrate physiological processes, in particular skeletal development and tissue mineralization. However, there is a lack of well characterized in vitro cell systems derived from fish calcified tissues. We describe here a protocol that was successfully used to develop the first calcified tissue-derived cell cultures of fish origin. Vertebra and branchial arches collected from young gilthead seabreams were fragmented then submitted to the combined action of collagenase and trypsin to efficiently release cells embedded in the collagenous extracellular matrix. Primary cultures were maintained under standard conditions and spontaneously transformed to form continuous cell lines suitable for studying mechanisms of tissue mineralization in seabream. This simple and inexpensive protocol is also applicable to other calcified tissues and species by adjusting parameters to each particular case.     

Identification of genetic loci affecting the establishment and development of Echinococcus multilocularis larvae in mice

Alveolar echinococcosis (AE) is a severe hepatic disorder caused by larval infection by the fox tapeworm Echinococcus multilocularis. The course of parasitic development and host reactions are known to vary significantly among host species, and even among different inbred strains of mice. As reported previously, after oral administration of parasite eggs, DBA/2 (D2) mice showed a higher rate of cyst establishment and more advanced protoscolex development in the liver than C57BL/6 (B6) mice. These findings strongly suggest that the outcome of AE is affected by host genetic factor(s). In the present study, the genetic basis of such strain-specific differences in susceptibility/resistance to AE in murine models was studied by whole-genome scanning for quantitative trait loci (QTLs) using a backcross of (B6 × D2)F₁ and D2 mice with varying susceptibility to E. multilocularis infection. For cyst establishment, genome linkage analysis identified one suggestive and one significant QTL on chromosomes (Chrs.) 9 and 6, respectively, whereas for protoscolex development, two suggestive and one highly significant QTLs were detected on Chrs. 6, 17 and 1, respectively. Our QTL analyses using murine AE models revealed that multiple genetic factors regulated host susceptibility/resistance to E. multilocularis infection. Moreover, our findings show that establishment of the parasite cysts in the liver is affected by QTLs that are distinct from those associated with the subsequent protoscolex development of the parasite, indicating that different host factors are involved in the host–parasite interplay at each developmental stage of the larval parasite. Further identification of responsible genes located on the identified QTLs could lead to the development of effective disease prevention and control strategies, including an intensive screening and clinical follow-up of genetically high-risk groups for AE infection.     

A novel approach to the establishment of dual cultures of pearl millet and Sclerospora graminicola

Dual cultures were successfully established using malformed florets of pearl millet infected with Sclerospora graminicola, the downy mildew pathogen. A higher proportion (86%) of calli from malformed florets formed dual cultures on Murashige and Skoog's (MS) medium with 2 mg 1^-1 of 2,4-dichlorophenoxy acetic acid (2,4-d), compared to shoot tips (25%). Fungal mycelium covered the entire surface of the callus within 30 days of placement of explants on the MS medium with 2 mg 1^-1 of 2,4-d. The infected calli also differentiated and produced plantlets when transferred to MS medium without 2,4-d.     

Factors affecting the establishment and maintenance of ‘Titan’ red raspberry root organ cultures

Root organ cultures of red raspberry (Rubus ideaus cv. Titan) were established from two cm long terminal segments of adventitious roots induced in vitro on micropropagated shoot cultures. Growth of root organ cultures was almost entirely by initiation and elongation of lateral roots rather than by terminal elongation from the apical meristem of the original root explants. Indolebutyric acid (IBA) was required for lateral root initiation and elongation. The optimal IBA concentration for lateral root initiation decreased from 0.5 to 0.1 mg/L from the first to the fifth passage in culture. Two cm root explants initiated more lateral roots per unit length than four cm explants. Liquid Anderson's medium was superior to other basal nutrient and vitamin formulations tested. Root growth in liquid culture was stimulated by aeration. Root organ cultures also grew on media solidified with gelrite or agar. A rapid transfer technique was developed for subculturing these which involved cutting and transferring 1 cm discs of roots and underlying medium. Adventitious bud formation occurred spontaneously, but sporadically in liquid cultures, and was not influenced by cytokinin, auxin (spat) light, or chilling.     

Changing arabinosylation patterns of wall-bound hydroxyproline in bean cell cultures

The arabinosylation patterns of wall-bound hydroxyproline in Phaseolus vulgaris L. cell suspension cultures were determined by separating free hydroxyproline and hydroxyproline-arabinose oligomers over a Bio-Gel P-2 column. Total hydroxyproline accounted for about 3.3% of wall dry weight during all growth phases of batch-cultured bean cells. The chemical arabinosylation patterns of wall-bound hydroxyproline varied during the lag phase and early log phase of the culture. First, an increase in nonglycosylated hydroxyproline occurred accompanied by a corresponding decrease in hydroxyproline tetra-arabinoside. During the early log phase the reverse happened. In later stages of growth the chemical arabinosylation patterns remained constant. The radiochemical arabinosylation patterns were also determined, after pulselabeling the cultures with [¹⁴C]proline at various times during growth, to be able to distinguish recently incorporated hydroxyproline. The time course of the arabinosylation pattern of this fraction indicated that the initial changes in the chemical pattern were due to the temporary incorporation of less extensively glycosylated hydroxyproline-containing protein into the cell wall.Abbreviations Hyp hydroxyproline - HA_n hydroxyproline arabinoside - with n arabinosyl residues - TFA trifluoroacetic acid     

亮斑扁角水虻幼虫酶解制备蛋白胨工艺优化及蛋白胨性质

【背景】用于餐厨垃圾等有机废弃物处理的亮斑扁角水虻(Hermetia illucens, HI)含有丰富的氨基酸和营养物质,是一种优质、经济的蛋白来源。【目的】利用亮斑扁角水虻幼虫(Hermetia illucens larvae, HIL)制备蛋白胨用于细菌培养,为亮斑扁角水虻的应用方式提供新的思路。【方法】以亮斑扁角水虻幼虫为原料,利用单因素试验确定最佳酶解条件,对比分析HIL蛋白胨和市售胰蛋白胨的基本性质和功能性状,并进行细菌培养、生物化学试验,利用两种蛋白胨分别培养大肠杆菌(Escherichia coli) ATCC 25922模式细菌并通过生长动力学分析评价应用效果。【结果】制备HIL蛋白胨的最佳酶解工艺为按1.3%(质量分数)添加复合酶(胰酶:碱性蛋白酶质量比为1:1),在pH 7.0、54℃条件下反应4 h,此时HIL的水解度为(19.34±0.15)%。HIL蛋白胨和市售胰蛋白胨的功能性状和生物化学试验差异不显著(P>0.05)。大肠杆菌ATCC25922在HIL蛋白胨培养基中生长的X_max和λ分别为6.44和2.45,在市售胰蛋白胨生长的...     

Electrophysiological investigation of the amino acid carrier selectivity in epithelial cells fromXenopus embryo

Summary The electrical responses induced by external applications of neutral amino acids were used to determine whether different carriers are expressed in the membrane of embryonic epithelial cells ofXenopus laevis. Competition experiments were performed under voltage-clamp conditions at constant membrane potential.Gly,l-Ala,l-Pro,l-Ser,l-Asn andl-Gln generate electrical responses with similar apparent kinetic constants and compete for the same carrier. They are [Na] _o and voltage-dependent, insensitive to variations in [Cl] _o and [HCO₃] _o , inhibited by pH _o changes, by amiloride and, for a large fraction of the current, by MeAIB. The increase in [K] _o at constant and negative membrane potential reduces the response, whereas lowering [K] _o augments it. l-Leu,l-Phe andl-Pro appear to compete for another carrier. They generate electrogenic responses insensitive to amiloride and MeAIB, as well as to alterations of membrane potential, [Na] _o and [K] _o . Lowering [Cl] _o decreases their size, whereas increasing [HCO₃] _o at neutral pH _o increases it.It is concluded that at least two and possibly three transport systems (A, ASC and L) are expressed in the membrane of the embryonic cells studied. An unexpected electrogenic character of the L system is revealed by the present study and seems to be indirectly linked to the transport function. l-Pro seems to be transported by system A or ASC in the presence of Na and by system L in the absence of Na. MeAIB induces an inward current.     

Analysis of rat adrenal cells in primary culture separated by velocity sedimentation

Summary Cell separation was used to follow the fate of the cortical cells of the adrenal gland in primary culture, and to assess some of the changes that occur as cells adapt to culture conditions. Primary cultures of rat adrenal gland were dissociated with trypsin and separated by velocity sedimentation at unit gravity. After two days in culture, cells showed a reproducible sedimentation profile consisting of two classes of cells with mean sedimentation rates of 5.8 and 2.1 mm/h, and a third sedimentation peak consisting mainly of nuclei at 0.5mm/h. All populations continued to incorporate ³H-thymidine in relatively constant proportion throughout the culture period, but the relative number of cells in the 2.1 mm/h peak increased two-fold in the last few days of primary culture. Cells labelled in primary culture, but separated after an additional 5 days in secondary culture had lost proportionately more labelled cells from the 5.8 mm peak. The results suggest that cells of the 2.1 mm peak survive longer in culture in a post-replicative condition.Work reported in this paper was performed while the author was a Research Fellow of the National Cancer Institute of Canada in the laboratory of Dr. N. Auersperg, Cancer Research Centre, University of British Columbia, Vancouver, B.C., Canada     

Cell-type-specific level of DNA nucleotide excision repair in primary human mammary and ovarian epithelial cell cultures

DNA repair, a fundamental function of cellular metabolism, has long been presumed to be constitutive and equivalent in all cells. However, we have previously shown that normal levels of nucleotide excision repair (NER) can vary by 20-fold in a tissue-specific pattern. We have now successfully established primary cultures of normal ovarian tissue from seven women by using a novel culture system originally developed for breast epithelial cells. Epithelial cells in these cultures aggregated to form three-dimensional structures called "attached ovarian epispheres". The availability of these actively proliferating cell cultures allowed us to measure NER functionally and quantitatively by the unscheduled DNA synthesis (UDS) assay, a clinical test used to diagnose constitutive deficiencies in NER capacity. We determined that ovarian epithelial cells manifested an intermediate level of NER capacity in humans, viz., only 25% of that of foreskin fibroblasts, but still 2.5-fold higher than that of peripheral blood lymphocytes. This level of DNA repair capacity was indistinguishable from that of normal breast epithelial cells, suggesting that it might be characteristic of the epithelial cell type. Similar levels of NER activity were observed in cultures established from a disease-free known carrier of a BRCA1 truncation mutation, consistent with previous normal results shown in breast epithelium and blood lymphocytes. These results establish that at least three "normal" levels of such DNA repair occur in human tissues, and that NER capacity is epigenetically regulated during cell differentiation and development.