Micropropagation of the medicinal plant, Scilla natalensis Planch.

Primary bulb explants of Scilla natalensis were cultured in vitro on modified MS medium. Some of these explants initiated shoots, which provided a sterile source of secondary leaf and bulb explants. The secondary explants responded similarly to various combinations of plant growth regulators. Shoots were initiated spontaneously on medium containing no plant growth regulators. The number of shoots initiated was increased by the addition of kinetin or thidiazuron (TDZ) alone, but was reduced by the addition of indole-3-acetic acid (IAA) or naphthalene acetic acid (NAA) alone. Optimal shoot initiation occurred on medium containing 1 to 2 mg l^–1 kinetin and 1 to 2 mg l^–1 IAA. These shoots were rooted on medium containing 1 mg l^–1 IAA. The plantlets were successfully acclimatised in the misthouse/shadehouse.     

Effect of 1,2-benzisoxazole-3-acetic acid on adventitious shoot regeneration and in vitro rooting in apple

 The effect of 1,2-benzisoxazole-3-acetic acid (BOA), compared to 1-naphthaleneacetic acid (NAA), on adventitious shoot formation in leaf portions and compared to indolebutyric acid (IBA), on in vitro rooting in the apple (Malus domestica Borkh) cultivars McIntosh and Gala, and one rootstock, Jork 9, was investigated. BOA at 43.0 μm or 2.7 μm at NAA in combination with 17.8 μm benzyladenine (BA), induced the highest number of explants to produce adventitious shoots in Jork 9. In Gala, the combination of 21.5 μm BOA with 1.0 μm thidiazuron (TDZ) or with 22.0 μm BA induced the highest regeneration percentages, 58 and 54%, respectively, giving more satisfactory results than NAA (where only 42% of leaf explants exhibited shoot formation). In McIntosh, the highest percentage of regeneration was obtained with 1.3 μm NAA and 22.0 μm BA, while 51% was the highest response obtained with the BOA treatment. The combination of BOA with TDZ completely inhibited regeneration activity in leaf portions of this cultivar. The shoots of all the genotypes obtained with the most morphogenetic NAA or BOA treatments were excised, multiplied and successfully rooted and hardened. The results demonstrate that the synthetic auxin BOA is active in inducing shoot regeneration from leaf explants of apple and that the activity of BOA in plant regeneration is genotype dependent. When BOA was used to induce rooting in apple microcuttings, lower rooting percentages were obtained than with IBA, showing that the effect of BOA in inducing root formation is very low and that it cannot be used routinely to replace IBA in the in vitro rooting of microcuttings. Received: 18 June 1998 / Revision received: 4 January 1999 / Accepted: 29 January 1999     

Optimization of biotin and thiamine requirements for somatic embryogenesis of date palm (Phoenix dactylifera L.)

Summary This study was conducted to examine the effect of biotin and thiamine concentrations on callus growth and somatic embryogenesis of date palm (Phoenix dactylifera L.). Embryogenic callus derived from offshoot tip explants was cultured on hormone-free MS medium containing biotin at 0, 0.1, 1, or 2 mg l⁻¹ combined with thiamine at 0.1, 0.5, 2, or 5 mg l⁻¹. Embryogenic callus weight, number of resultant embryos, and embryo length were significantly influenced by thiamine and biotin concentration. The optimum callus growth treatment consisted of 0.5 mg l⁻¹ thiamine and 2 mg l⁻¹ biotin. This treatment also gave the highest number of embryos. Embryo elongation was greatest at 0.5 or 2 mg l⁻¹ thiamine combined with 1 mg l⁻¹ biotin. Embryos from all treatments germinated and regenerants exhibited normal growth in soil. This study provides an insight into the importance of optimizing various culture medium components to overcome in vitro recalcitrace of date palm.     

A Novel Three-dimensional Flow Chamber Device to Study Chemokine-directed Extravasation of Cells Circulating under Physiological Flow Conditions

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell homing and tumor metastasis. The three-dimensional flow chamber device (hereafter the 3D device) is a novel in vitro technology that recreates physiological shear stress and allows each step of the cell extravasation cascade to be quantified. The 3D device consists of an upper compartment in which the cells of interest circulate under shear stress, and a lower compartment of static wells that contain the chemoattractants of interest. The two compartments are separated by porous inserts coated with a monolayer of endothelial cells (EC). An optional second insert with microenvironmental cells of interest can be placed immediately beneath the EC layer. A gas exchange unit allows the optimal CO₂ tension to be maintained and provides an access point to add or withdraw cells or compounds during the experiment. The test cells circulate in the upper compartment at the desired shear stress (flow rate) controlled by a peristaltic pump. At the end of the experiment, the circulating and migrated cells are collected for further analyses. The 3D device can be used to examine cell rolling on and adhesion to EC under shear stress, transmigration in response to chemokine gradients, resistance to shear stress, cluster formation, and cell survival. In addition, the optional second insert allows the effects of crosstalk between EC and microenvironmental cells to be examined. The translational applications of the 3D device include testing of drug candidates that target cell migration and predicting the in vivo behavior of cells after intravenous injection. Thus, the novel 3D device is a versatile and inexpensive tool to study the molecular mechanisms that mediate cellular extravasation.     

Tissue-Characteristic Expression of Mouse Proteome

The mouse is a valuable model organism for biomedical research. Here, we established a comprehensive spectral library and the data-independent acquisition–based quantitative proteome maps for 41 mouse organs, including some rarely reported organs such as the cornea, retina, and nine paired organs. The mouse spectral library contained 178,304 peptides from 12,320 proteins, including 1678 proteins not reported in previous mouse spectral libraries. Our data suggested that organs from the nervous system and immune system expressed the most distinct proteome compared with other organs. We also found characteristic protein expression of immune-privileged organs, which may help understanding possible immune rejection after organ transplantation. Each tissue type expressed characteristic high-abundance proteins related to its physiological functions. We also uncovered some tissue-specific proteins which have not been reported previously. The testis expressed highest number of tissue-specific proteins. By comparison of nine paired organs including kidneys, testes, and adrenal glands, we found left organs exhibited higher levels of antioxidant enzymes. We also observed expression asymmetry for proteins related to the apoptotic process, tumor suppression, and organ functions between the left and right sides. This study provides a comprehensive spectral library and a quantitative proteome resource for mouse studies.     

Formation and preservation of cortical layers in slice cultures

During cortical development, neurons generated at the same time in the ventricular zone migrate out into the cortical plate and form a cortical layer (Berry and Eayrs, 1963, Nature 197:984–985; Berry and Rogers, 1965, J. Anat. 99:691–709). We have been studying both the formation and maintenance of cortical layers in slice cultures from rat cortex. The bromodexyuridine (BrdU) method was used to label cortical neurons on their birthday in vivo. When slice cultures were prepared from animals at different embryonic and postnatal ages, all cortical layers that have already been established in vivo remained preserved for several weeks in vitro. In slice cultures prepared during migration in the cortex, cells contiuned to migrate towards the pial side of the cortical slice, however, migration ceased after about 1 week in culture. Thus, cortical cells reached their final laminar position only in slice cultures from postnatal animals, whereas in embryonic slices, migrating cells became scattered throughout the cortex. Previous studies demonstrated that radial glia fibers are the major substrate for migrating neurons (Rakic, 1972, J. Comp. Neurol. 145:61–84; Hatten and Mason, 1990, Experientia 46:907–916). Using antibodies directed against the intermediate filament Vimentin, radial glial cells were detected in all slice cutures where cell migration did occur. Comparable to the glia development in vivo, radial glial fibers disappeared and astrocytes containing the glia fibrillary-associated protein (GFAP) differentiated in slice cultures from postnatal cortex, after the neurons have completed their migration. In contrast, radial glial cells were detected over the whole culture period, and very few astrocytes differentiated in embryonic slices, where cortical neurons failed to finish their migration. The results of this study indicate that the local environment is sufficient to sustain the layered organization of the cortex and support the migration of cortical neurons. In addition, our results reveal a close relationship between cell migration and the developmental status of glial cells. © 1992 John Wiley & Sons, Inc.     

Initiation and characterization of five embryonic cell lines from the cotton boll weevil anthonomus grandis in a commercial serum- free medium

Summary Five continuous cell lines were initiated from embryonic tissue of the cotton boll weevilAnthonomus grandis Boheman in a commercially available, serum-free medium (Excell 401) and have undergone in excess of 60 passages. Isoenzyme analysis confirmed that the lines originated from boll weevil tissue. Four of the lines grew as single attached cells of either epithelioid or fibroblastoid morphology. The fifth line, BRL-AG-2, grew primarily as cell aggregates and was found to release ecdysteroids (primarily ecdysone) into the culture medium. Evidence was also obtained suggesting that line BRL-AG-2 synthesizes chitin. Three lines, BRL-AG-1, BRL-AG-3A, and BRL-AG-3C, could be induced to produce an antibacterial factor(s) which was released into the culture medium.     

EGF and TGFα modulate structural and functional differentiation of the mammary gland from pregnant mice in vitro: Possible role of the arachidonic acid pathway

Epidermal growth factor (EGF) has been suggested to be involved in mammary gland development by mitogenic stimulation of the ductal and alveolar epithelium in virgin mice. The present studies demonstrate that also in late-pregnant mice EGF leads to proliferation of the ductal, ductular, and alveolar epithelium. The mitogenic effect is associated with structural and functional dedifferentiation of alveolar cells as revealed by analysis of morphology, expression of cytosolic and secretory proteins, and fatty acid synthesis. Using a combination of metabolic inhibitors, the dedifferentiating effect of EGF could be blocked while the mitogenic action was not influenced. This finding demonstrates that the signal transduction pathway leading to dedifferentiation and mitosis can be separated, and that the dedifferentiating effect of EGF is independent of its mitogenic properties, but is probably mediated by activation of the arachidonic acid-dependent pathways (cyclo- and lipoxygenase pathways). Release of arachidonic acid from the endogenous phospholipid pool was found to be an early response of the explants to EGF. Accordingly, arachidonic acid itself proved to be capable of inducing epithelial dedifferentiation but failed to stimulate proliferation. TGFα showed qualitatively similar effects as EGF but was generally a stronger agonist. It is suggested that EGF and TGFα also play a role in mammary gland physiology during pregnancy by final developing and maintanance of the lobulo-alveolar structure in the mammary gland and prevention of premature onset of lactation, and that this is mediated through the PLA₂-arachidonic acid signalling cascade.