Obtaining Suspensions of Animal Cells in Metaphase from Cultures Propagated on Glass

Cells of some strains continuously cultivated in vitro have a pronounced tendency to lose their attachment to glass during mitosis. If division of these cells is arrested in meta-phase by treatment with colchicine, many of the cells are set free and float into the medium. Advantage has been taken of this property to collect large numbers of suspended cells in c-metaphase. By applying standard procedures of staining with orcein and manual squashing to these cells, preparations are obtained that are useful for critical chromosome analysis. The procedure consists in treatment of cultures with colchicine (final concentration 0.1 μg/ml) for 18-24 hr; agitation in hypotonic medium (quarter-strength Tyrode's or CMRL-1066) for 5 min; addition of 1:3 acetic-alcohol for 5-10 min; removal and centrifugation of the resulting fluid mixture from the culture; and resuspension of the sedimented cells in a small volume of the fluid. Advantages of the method are: large numbers of metaphase plates can be examined on a single slide, the chromosomes are contracted and well separated, and cytoplasmic boundaries are preserved.     

Subcortical Rotation and Specification of the Dorsoventral Axis in Newt Eggs

The specification of the dorsoventral axis in naturally polyspermic eggs of the Japanese newt, Cynops pyrrhogaster , was first examined by studies on the spatial relationship between the dorsal midline of the future body plan and the sperm entrance points (SEPs ¹ ). On local insemination, the dorsal blastopore lip was usually found to be formed opposite the SEPs, as in anuran monospermic eggs. Next the movements of the subcortical layer and the cortex were analyzed. "Subcortical rotation" was observed, similar to that of Xenopus laevis eggs with respect to its timing and extent, and its direction was shown to predict the embryonic axis of the eggs. Thus, the dorsoventral axis was concluded to be determined by essentially the same mechanism in the newt as in Xenopus .
Owing to their large size and long first cell cycle, newt eggs appear to be suitable material for study of subcortical rotation, but their behavior is unique in that subcortical rotation occurs in only the vegetal hemisphere so that the subcortical layer stretches in the future dorsal side. Studies on the movement of Nile blue spots suggested that the cytoplasm under the cortex in newt eggs consists of two layers.     

Interactions among Cells of the Developing Neural Retina in vitro1

Two models are presented for mechanisms leading to the formationof layers during the development of the chick neural retina.The first proposes that the different cells possessintrinsiccharacteristics that allow them to self sort into specific patterns.The second suggests that the cells migrate, or are moved, alonga substrate which guides them into specific locations. Supportingevidence is presented for both hypotheses, suggesting that theoverall process is highly complex, using elements of each mechanism.     

Two Improved Methods for Obtaining Axenic Cultures of Cyanobacteria

Scoring the agar plate before incubation under unidirectional light led to a rapid separation of gliding filamentous cyanobacteria from their contaminating bacteria. Twenty strains were purified by the method. Additionally, 13 axenic cyanobacterial strains were isolated from pour plates made after treatment of cyanobacterial cultures in tryptone-yeast extract-glucose broth with cycloserine in darkness to select for obligate photoautotrophs.     

An Organotypic Culture of the Newt Retina together with Other Tissues of the Posterior Eye Segment as a Model for Studying the Effects of Cell Adhesion Glycoproteins

The adult newt retina explanted together with the posterior eye wall and cultivated for a short time in a serum-free medium was tested as an experimental model by several criteria, including the expression of protein markers of the main retinal cell types. Some differences in the expression of specific photoreceptor, interneuron, and glial cell proteins as well as the localization of acetylcholinesterase activity were found during in vitro cultivation. Using this model, preliminary tests of new cell adhesion glycoproteins from the bovine retina and pigment epithelium were conducted, and the role of pigment epithelial cell proteins in improving cell viability in the cultivated newt retina was revealed. Moreover, the fraction of basic adhesion proteins from the bovine pigment epithelium improved the survival potential of the macroglial (Muller) cell population, compared to that in the control.     

Improved Enrichment and Isolation Procedures for Obtaining Pure Cultures of Beggiatoa

Scoring agar surfaces with alginate swabs before placing washed filaments of Beggiatoa on the agar has greatly increased the rate at which single filaments move from contaminated areas. Numerous morphological types of pure cultures have been grown in organic media supplemented with either catalase or reducing agents. Aerated sewage was used as the enrichment source.     

An Immunohistochemical Study of Localization of the Calcium-Binding Protein Recoverin in Retina of the Newt Pleurodeles waltl

The presence and localization of the calcium-binding protein recoverin, initially found in photoreceptors of the bovine eye, were immunochemically studied in retina of the new Pleurodeles waltl. Polyclonal monospecific antibodies against recoverin were raised and the methods of immunoblotting and indirect immunofluorescence were used. A protein with an apparent molecular mass of 26 kDa was found in the retina extract, which was specifically stained by the antibodies against recoverin. Localization of recoverin was studied on the retina sections: an intense reaction was found in the inner segments and a weak reaction was found in the basal part of the outer segments of photoreceptors and in Landolt's clubs of displaced bipolars. The results we obtained suggest for the first time the presence of recoverin in the retina of a representative of the Urodeles and indicate to interspecific conservativeness of this protein and differences of its localization in the retina photoreceptors in different species. The data obtained open a possibility of using recoverin as a marker protein of photoreceptors and displaced bipolars in studies of retina regeneration in newts.     

A Yolk-granule Component Acts as an Adhesive Material for Dissociated Gastrula Cells of the Newt, Cynops pyrrhogaster

Yolk granules were collected from full-grown ovarian oocytes of the newt, Cynops pyrrhogaster , and dissolved in 3% acetic acid or 8 M urea solution. Culture dishes were then coated with either of the yolk-granule solutions. The yolk-coated surfaces acted as adhesive substrata for dissociated gastrula cells, which showed active locomotion when spread on the surfaces. Divalent cation was required for cell adhesion and spreading on the yolk-coated surface. Trypsin and glycosidase digestions of dissociated cells or the yolk-coated surfaces inhibited cell adhesion and spreading. Addition of concanavalin A to the culture medium inhibited cell spreading on the yolk-coated surfaces, while high concentration (50 mM) of the saccharides, D-galactose, D-lactose and D-mannose, had a slightly inhibitory effect on cell spreading.
Two yolk components (30-kD and 108-kD proteins) were isolated from yolk granules and applied to the culture dish surfaces. Surfaces coated with the 30-kD protein showed strong adhesiveness for dissociated gastrula cells.     

Commentary Salicylate Trapping of -OH as a Tool for Studying Post-Ischemic Oxidative Injury in the Isolated Rat Heart

The use of salicylate as a chemical trap for -OH represents a simple and convenient alternative to the use of spin trapping techniques to study oxidative injury in isolated perfused organs. In these systems, salicylate is included in the perfusion buffer at concentrations ranging from 0.1 to 2mM depending on the detection apparatus employed. In our studies, we have used a coulometric detector, which has a theoretical efficiency of 100% as compared to 1-5% for the standard glassy carbon electrode. We have been able to generate reproducible results by inclusion of only 100 μM salicylate, a concentration demonstrated not to affect pre- or post-ischemic cardiac function. In initial studies, we observed an increase in perfusate 2,5-dihydroxybenzoic acid consistent with an early post-ischemic burst of -OH, not unlike that reported using spin trapping techniques. Since then we and others have used this technique to examine possible relationships between -OH formation and treatments that alter post-ischemic cardiac functional recovery. For example, preischemic loading of hearts with copper results in increases in postischemic dysfunction and LDH release that were associated with an increase in 2,5-dihydroxybenzoate and by inference, -OH formation. Alternatively, we have reported that the nitroxide spin label, TEMPO, reputed to be a superoxide dismutase mimetic, decreased post-ischemic arrhythmias and 2,5-dihydroxybenzoate formation. Most recently, we have observed that preischemic loading of hearts with zinc-bis-histidinate results in improved post-ischemic cardiac function and decreased LDH release; changes that were associated with decreased 2,5-dihydroxybenzoate formation. These studies indicate that under certain conditions, salicylate is a valuable alternative to spin trapping techniques to probe the role of -OH in cardiac oxidative injury, particularly when applied to the isolated perfused heart preparation.     

Ultrastructural Observations on Proliferating Storage Cells of Mature Cotyledons of Phaseolus vulgaris L. Cultured in vitro

Explants of mature cotyledons of Phaseolus vulgaris form callusrapidly when cultured in vitro with their adaxial surfaces embeddedin a solidified nutrient medium containing coconut milk, kinetinand 2,4-D. Proliferation is confined to the highly polyploidstorage cells and commences near the adaxial epidermis, whichis soon ruptured by the callus developing internally. Callusformation progresses to the abaxial tissue and within 3–4weeks sub-culturing is possible. The in vitro grown storage cells undergo thinning of their walls,loss of food reserves, hypertrophy, development of various new-wallsand nuclear activation leading to division. The induction ofnuclear and cell divisions within this mature storage tissuecontrasts with normal germination in which these cells undergorapid senescence after depletion of their food reserves. Nuclear division in early callus growth is apparently mainlyamitotic. It is preceded by the development of multiple nucleoli.The nuclear envelope also becomes more complex and deeply lobed;leading to formation of a nuclear isthmus and final separationinto two nuclei. No chromosomes are visible during nuclear fragmentation.Amitosis is accompanied by freely-forming walls, which may developadjacent to a nuclear isthmus and perhaps participate in nuclearfragmentation. Large labyrinthine wall bodies frequently occuron these walls. Mitoses are only observed in already dividedstorage cells. A cell plate forms between the two daughter nuclei,and microtubules are present at its margins in contrast to freely-formingwalls where none are evident. Phaseolus vulgaris L., bean, in-vitro culture, cotyledon, ultrastructure     

Expression of Neuronal Specificities in "Transdifferentiating" Cultures of Neural Retina

Cells dissociated from the neural retina of embryonic chick differentiate into lens and pigment cells, when cultured in vitro. Using 3.5-day-old and 8.5-day-old chick embryos, we examined whether neuronal specificities would be expressed in such transdifferentiating cultures of neural retinal cells. The synthesis of acetylcholine and γ-aminobutyric acid (GABA) and the activity of choline acetyl transferase (CAT) was searched for in these cultures. The synthesis of an appreciable amount of these two putative neurotransmitters was detected in cultures of 3.5-day-old embryonic retinas by about 15 days. The activity of CAT was maximum in 7-day cultures of the 3.5-day-old materials and in 2-day cultures of the 8.5-day-old materials, and then decreased. Concomitant with the decrease of CAT-activity, δ-crystallin became detectable and increased thereafter. CAT-activity changed in parallel with the increase in the number of small neuroblast-like cells in cultures. The results demonstrate that the neuronal specificity identified by the appearance of acetylcholine and GABA and of the enzyme for the synthesis of acetylcholine is expressed in the early period of transdifferentiating cultures, which would later differentiate into lens and pigment cells. The possible mechanisms of the transition from neuronal to non-neuroretinal specificities of the transdifferentiating cultures are discussed.     

Plasmolysis as a Tool to Reveal Lead Localization in the Apoplast of Root Cells

In order to analyze the distribution of lead between cell walls and plasmalemma, two-day-old maize seedlings (Zea mays L.) were incubated for 24 h on a solution of lead nitrate at a concentration causing 50% inhibition of root growth (10^–5 M). Using the histochemical technique (precipitation of lead dithizonate), the distribution of lead in plasmolyzed and nonplasmolyzed cells of the root cortex was compared. This allowed us to separate the lead bound by cell walls from the lead located on the protoplast surface and in the periplasmic space. The plasmolysis was conducted prior to histochemical reaction by the incubation of seedling roots in 0.6 M sucrose solution for 30 min. The lead precipitates were located in cell walls and on the surface of protoplast. A small amount of lead was found in periplasmic space of some cells in root cortex. It is suggested that the lead is bound not only to the cell wall matrix but also to the plasmalemma.     

In vitro Decline in Plant Cultures: Detection of a Legion of Covert Bacteria as the Cause for Degeneration of Long-term Micropropagated Triploid Watermelon Cultures

In vitro cultures of triploid seedless watermelon cv. Arka Manik displayed a decline in shoot and root growth after 4–5 years of active culturing. Visibly clean cultures upon indexing on enriched media showed covert bacteria, and a significant improvement in proliferation and rooting in response to surface sterilization. The bacteria however survived endophytically. Low pH and reduced clarity of agar gelled medium were found to mask the expression of bacteria in the tissue culture medium. Gentamycin, streptomycin or broad-spectrum bactericide cefazolin provided as a 2 ml overlay in the medium in factorial combination at 0 or 50 mg l^–1 resulted in selective suppression of some bacteria depending on the treatment and eight bacterial clones comprising of four Gram-positive (Bacillus spp.) and four-Gram negative (3 × Pseudomonas spp. and 1 × Aeromonas sp.) strains were isolated from the cultures. Provision of 50 mg l^–1 gentamycin in 2 ml overlay in the multiplication or rooting medium coupled with occasional decontamination of cultures helped in circumventing the decline problem. The plants established in the field after 6 years of active in vitro culturing appeared normal and fertile suggesting the feasibility of keeping cultures for long periods, thus saving time and other resources. Freeing the cultures from covert bacteria was complicated by the presence of different bacterial types and this will be addressed later.     

A System for Culturing Iris Pigment Epithelial Cells to Study Lens Regeneration in Newt

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, heart, the jaw ¹. Specifically, newts are unique for its lens regeneration capability. Upon lens removal, IPE cells of the dorsal iris transdifferentiate to lens cells and eventually form a new lens in about a month ^2,3. This property of regeneration is never exhibited by the ventral iris cells. The regeneration potential of the iris cells can be studied by making transplants of the in vitro cultured IPE cells. For the culture, the dorsal and ventral iris cells are first isolated from the eye and cultured separately for a time period of 2 weeks (Figure 1). These cultured cells are reaggregated and implanted back to the newt eye. Past studies have shown that the dorsal reaggregate maintains its lens forming capacity whereas the ventral aggregate does not form a lens, recapitulating, thus the in vivo process (Figure 2) ^4,5. This system of determining regeneration potential of dorsal and ventral iris cells is very useful in studying the role of genes and proteins involved in lens regeneration.     

Effects of a Carcinogen, N-methyl-N'-nitro-N-nitrosoguanidine on Blastema Cells and Blastema Formation in Newt Limb Regeneration

The effects of a potent, carcinogen, N -methyl- N' -nitro- N -nitrosoguanidine (MNNG) on blastema formation and blastema cells were studied in the adult newt by means of scanning electron microscopy. By administration of MNNG to the blastema of the limbs the formation of the basement lamella, which lies between the epithelium and the mesenchyme, was effectively inhibited at least for a month. Basement lamella in treated limbs is formed 30 or 40 days after carcinogen administration. MNNG altered significantly cell surface morphology and cell motility of blastema cells isolated in vitro. Membrane's specializations such as the formation of filopodia were inhibited. Motility of the treated cells was much reduced compared to the control. The role of these alterations in the carcinogen-induced abnormal regeneration is discussed.