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Ultrastructural immunogold labelling of human Langerhans cells enriched epidermal cell suspension.

Colloidal gold particles are well suited as markers in electron microscopy. Indirect immunogold staining was used to identify cell membrane antigens defined by monoclonal antibodies OKT6 and BL6 on human Langerhans cells (LC) in suspensions. Isolated epidermal cells were obtained by skin trypsinization and enriched or depleted in OKT6 positive on BL6 positive LC using the panning method: incubation of OKT6 or BL6 preincubated cells on immunoglobulin coated dishes. Indirect immunogold staining was then performed after prefixation in 2% paraformaldehyde. In LC enriched suspensions, only LC exhibited a specific membrane labelling with OKT6 or BL6 recognized by the presence of small evently distributed gold granules. Neither Birbeck granules, nor other cytoplasmic organelles, were labelled. No other epidermal cells were found positive. In LC depleted suspensions, no labelling was observed. Immunogold labelling on LC enriched suspensions after panning is now in progress for the qualitative evaluation and the quantitative analysis of cell surface constituents and antigens expressed by human dendritic epidermal cells.

Antibodies, Monoclonal↗

Studies on plating efficiency and estimation of viability of suspensions of Paracoccidioides brasiliensis yeast cells.

Mild sonication was used to obtain single cell suspensions of Paracoccidioides brasiliensis. These cells were intact by microscopic criteria. Direct cell counts in a given inoculum and colony formation on various media were used to determine plating efficiency. Sonicated and nonsonicated cell suspensions were used to study plating efficiency and to estimated viability by means of vital dyes. Methylene blue Erythrosin B, and Janus green were unreliable when used with P. brasiliensis, but vital dyes were accurate when tested with Candida albicans. Acridine orange gave more meaningful results of viability. Estimates of viability, however, changed significantly as a result of relatively minor alterations in the composition of the suspending medium. In initial experiments, the plating efficiency of P. brasiliensis was dismally low. It descended abruptly with increasing dilution of inoculum. Efficiency was much improved if horse serum was added to brain heart infusion plates or if glucose glycine yeast extract (GGY) plates were incubated at room temperature and mycelial colonies were counted. With the technique we report, current plating efficiency of sonicated suspensions is of the order of 25%. Our results and procedures have an important bearing upon those studies concerned with in vitro killing of P. brasiliensis in suspensions or with isolating this fungus from clinical or environmental specimens.

Acridine Orange↗

Arrest of developmental conversion of type II to type I fibres after suspension hypokinesia.

The effects of hypokinesia and of the lack of gravity on muscle fibres, fibre type composition and myosin light chain pattern, as well as on muscle mechanoreceptors were investigated in the slow-twitch soleus (SOL) and fast-twitch extensor digitorum longus (EDL) muscles of young growing and adult rats after suspension hypokinesia (SH) of their hind limbs. The animals were suspended by their tail so that their hind limbs were relieved of their normal weight-bearing function for 3-6 weeks. In normal 3- to 4-week-old rats the SOL contained about 50% type I fibres and their percentage increased up to about 80% until the 10th week, with simultaneous reduction of type IIA fibres. After 3 to 6 weeks of suspension treatment maintained from 3- to 4-week-old rats up to 6 to 10 weeks of age, the SOL still only contained about 50% of type I fibres. The content of fast LC1 and LC2 in the SOL of 6-week-old rats after 3 weeks of suspension was higher than that of control litter-mates reflecting the higher occurrence of IIA fibres in the suspended solei. No changes in fibre type composition were observed after SH performed in adult rats. SH thus leads, in young animals, to the arrest of conversion of type IIA to type I fibres resulting in the persistence of the fibre type composition and of the myosin light chain pattern corresponding to those present in the SOL at the time of the onset of suspension. In both young and adult rats, SH markedly decreased the mass and the mean cross-sectional area of the SOL, mainly due to the severe atrophy of type I fibres. We observed no signs indicating conversion of type I back to type IIA muscle fibres due to the SH either in young or adult animals. In contrast to profound changes in the SOL, no significant differences were found in the EDL in any of the parameters studied. No changes in the investigated parameters of muscle spindles and tendon organs were observed after SH, performed either in young or in adult rats. We thus conclude that SH leads to muscle atrophy and that it influences mainly or exclusively type I fibres in muscles with a postural function such as the SOL.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

Chinese hamster ovary cells cultured in low concentrations of fetal bovine serum: cloning efficiency, growth in suspension, and selection of drug-resistant mutant phenotypes.

Reducing serum concentrations in media provides a potential cost advantage. To determine whether such media could be used for applied mutagenesis assays, we measured cloning efficiency and growth parameters in suspension of Chinese hamster ovary cells cultured in reduced serum with or without additives (1 microgram/ml insulin, 3 X 10(-7) M linoleic acid, 1 X 10(-8) M H2SeO3) or bovine serum albumin (BSA, 1% wt/vol). With the additives and less than or equal to 0.5% fetal bovine serum (FBS), Ham's F12 medium (without hypoxanthine and thymidine) was more optimal than alpha Eagle's minimum essential medium (MEM) (without ribosides and deoxyribosides) for low density cloning and high density suspension growth. Acceptable cloning efficiencies were obtained with 2% FBS plus BSA without additives in either medium; the addition of BSA resulted in improved colony size and more compact colony morphology. In alpha MEM, satisfactory growth rates and maximum saturation densities in suspension culture were obtained only with 5% FBS; in Ham's F12, 1% FBS + deoxycytidine + BSA yielded satisfactory suspension growth. Spontaneous mutant frequencies were compared for each medium containing 10% dialyzed FBS (DFBS), 1% FBS plus BSA, or 2% FBS plus BSA. The spontaneous frequency of azaadenine-resistant phenotypes (mutant at the aprt locus) in 1% FBS plus BSA was significantly lower than the frequency observed in 2% FBS plus BSA or 10% DFBS. Frequencies of spontaneous mutants resistant to thioguanine (hgprt locus) or fluorodeoxyuridine (tk locus) were similar with 10% DFBS, 1% FBS plus BSA, or 2% FBS plus BSA. Compared to alpha MEM with 10% DFBS, frequencies of drug-resistant mutants induced by ethyl methanesulfonate or mitomycin C (MMC) were not significantly lower in alpha MEM with 2% FBS plus BSA; observed mutant frequencies induced by dimethylnitrosamine or benzo(a)pyrene seemed to be decreased at lower survival levels.

Animals↗

Application of some droplet sedimentation theories to layered erythrocyte suspensions.

The applicability of some existing droplet sedimentation theories to erythrocyte suspensions was investigated using glutaraldehyde-fixed erythrocytes from horse, canine, pig, chicken, and human. The Svensson criteria were shown to underestimate the load supportable by a density gradient. The available theories on droplet formation times could not predict each other, and the data on erythrocyte suspensions, especially at high suspension concentrations. It was finally argued that, since the behavior of erythrocytes was not controlled by the diffusion process, the erythrocyte suspensions were not expected to exhibit lasting or absolute stability even when the particle load was small.

Animals↗

Effects of suspension density on microbial metabolic processes.

Respiration of yeasts Saccharomyces cerevisiae, Rhodotorula glutinis, Endomyces magnusii, and Candida utilis, and of bacteria Escherichia coli and Bacillus megaterium, anaerobic production of CO2 by S. cerevisiae, active transport of quinovose by R. glutinis and of L-proline and L-leucine by S. cerevisiae were highly dependent on cell suspension density. Respiration of S. cerevisiae in the presence of glucose decreased in a biphasic fashion from 140 to 40 nmol O2 per mg dry solid per min as suspension density increased from 0.01 to 2 mg/mL. Higher partial pressures of oxygen further enhanced the trend. The active transports were affected monophasically in the maximum rate of uptake which was as much as ten times greater at low than at high suspension densities. A component of the external medium is suspected to cause the decrease of metabolic functions at higher cell densities, acting as a noncompetitive inhibitor. The component was present and mutually active in suspensions of the various yeasts as well as of bacteria. Its properties and results of model experiments suggest it to be dissolved carbon dioxide.

Anaerobiosis↗

Suture injury to the urinary tract in urethral suspension procedures for stress incontinence.

The case histories of women attending the Urogynecology Department at the Royal Women's Hospital and Mercy Hospital for Women were reviewed between 1986 and 1998 to determine the incidence and postoperative morbidity caused by suture injury to the urinary tract following urethral suspension surgery for stress incontinence. In our department 1103 Burch colposuspensions and 61 Stamey urethral suspensions have been performed. Intraoperative cystourethroscopy was performed routinely for the early detection and treatment of urinary tract injury. Intravesical sutures were found by routine intraoperative cystoscopy in 1 Stamey suspension, 1 open Burch colposuspension and 3 laparoscopic Burch colposuspensions. Ureteric suture ligation was diagnosed in 2 women intraoperatively and 1 women postoperatively after laparoscopic Burch colposuspension. Two women presented with late complications from intravesical sutures following open Burch colposuspension. A further 7 women referred with urinary symptoms were found to have intravesical sutures, 2 following Burch colposuspension, 4 following Stamey urethral suspension and 1 following the Marshall-Marchetti-Kranz procedure. Seven of the 9 women diagnosed with intravesical sutures presented with bladder or pelvic pain, frequency or urinary tract infection. Two women had recurrent stress incontinence and were found to have a intravesical suture on routine cystoscopy at the time of stress incontinence surgery. Suture removal, with any accompanying calculus, was achieved cystoscopically with almost immediate resolution of symptoms without loss of urinary control in all cases. Non-absorbable intravesical sutures occurring as a result of suture misplacement or erosion is an infrequent but important complication of stress incontinence surgery, but should be suspected if pain and irritative bladder symptoms or recurrent urinary infection occur postoperatively. Cystourethroscopy performed intra-operatively or postoperatively is essential for early diagnosis and treatment.

Adult↗

Interrelation between HeLa-S3 cell transfection and hemolysis in red blood cell suspension using pulsed ultrasound of various duty cycles.

We have studied the in vitro transfection of a plasmid DNA with the lacZ gene to HeLa-S3 cells and hemolysis in a red blood cell (RBC) suspension under pulsed ultrasound with duty cycles gamma of 10, 20 and 30% using a digital sonifier at a frequency of 20 kHz and an intensity of 6.2 W/cm(2) on the surface of a horn tip. Cultured HeLa-S3 cells in suspension were exposed to pulsed ultrasound for an apparent exposure time t' from 0 to 60 s. HeLa-S3 viability decreased as a single exponential function of the total exposure time t = gammat' with a common time constant tau = 3.8 s for three duty cycles. Transfection was evaluated by counting the number of beta-galactosidase(beta-Gal)-positive cells relative to the total number of cells. Pulsed ultrasound provided an enhanced transfer of the beta-Gal plasmid to HeLa-S3 cells, 3.4-fold as compared with that in the case of the control. The optimal transfection efficiencies were 0.75, 0.80 and 0.74% near t = tau with gamma = 10, 20 and 30%, respectively. The number ratio of beta-Gal-positive cells to the surviving cells after exposure increased with t' according to a modified logistic equation. The degree of hemolysis also increased exponentially with t' at a time constant tau' = tau(0)/gamma for the RBC suspension in physiological saline at a hematocrit concentration of 0.5% with tau(0) = 0.9 s. Thus the total exposure time for the optimal transfection efficiency was tau, that is, nearly four times of tau(0). Hemolysis in the RBC suspension may be a useful model for determining optimal transfection by pulsed ultrasound of various duty cycles.

Animals↗

Suture suspension platysmaplasty for neck rejuvenation revisited: technical fine points for improving outcomes.

BACKGROUND: The suture suspension platysmaplasty technique for neck rejuvenation was introduced more than 13 years ago. Over the years and after a large number of procedures performed for all classes of neck deformities, a number of technical fine points evolved that resulted in even more aesthetically pleasing and predictable results. These technical fine points are presented in detail, and their direct anatomic effects are analyzed. Finally, the points that constitute the youthful neck are redefined with detailed schematic presentation. ANATOMIC CONSIDERATIONS: All aspects of neck rejuvenation with the suture suspension platysmaplasty are analyzed, with a focus on the changes that occur in terms of the following six key anatomic points: cervicomental angle depth, mandibular border definition, mandibular angle definition, labiomandibular fold prominence (jowling), mental prominence, and neck width. TECHNICAL FINE POINTS: Additional technical fine points have evolved over the past 13 years to optimize the results and produce a more natural appearing and aesthetically pleasing neck: the "angle loop" of the suspension sutures, anatomic suction-assisted lipectomy using appropriate cannulas, application of moderate tension to the interlocking suspension suture to avoid the "overcorrected neck," imbrication of the midline platysma, conservative skin excision, and augmentation of the chin to increase the depth of the cervicomental angle. CONCLUSION: The interlocking suture technique, by creating an artificial permanent "ligament" under the mandible, corrects the anatomic components of the neck (points 1 to 6) with excellent long-term outcomes and patient satisfaction rates. The additional technical fine points have made the aesthetic results more natural and pleasing, while making the technique more simple, safe, and reproducible.

Female↗

Long-term results of suture suspension platysmaplasty for neck rejuvenation: a 13-year follow-up evaluation.

BACKGROUND: Neck rejuvenation has been one of the most important components in treatment for the aging face because the neck frequently is the first feature to show signs of aging. Several techniques have been described, but assessment of the long-term effects, although limited, has shown frequent recurrence of neck ptosis and the need for reoperation. The suture suspension technique was introduced 13 years ago. This article presents the long-term results for this technique, illustrating the different neck types, their classification, and their outcomes. METHODS: For this study, 100 patients who underwent neck rejuvenation using the suture suspension platysmaplasty technique were randomly selected. They represented the four patient groups examined: 5-year group (47 patients), 7-year group (28 patients), 10-year group (19 patients), and 13-year group (6 patients). The key anatomic points for each patient were evaluated preoperatively and postoperatively by five independent observers who analyzed the outcomes with a simplified scoring system (1 to 10 for each anatomic point). The scoring system was used for all the patient age groups and applied to all six key anatomic points of the neck: cervicomental angle depth, mandibular border definition, mandibular angle definition, labiomandibular fold prominence (jowling), mental prominence, and neck width. Patient satisfaction surveys were offered to all patients included in the study. RESULTS: All the patient groups showed significant aesthetic improvement (in terms of their scores) for all the anatomic points (1-6) evaluated. Specifically, the long-term effect of the procedure was the most impressive for the cervicomental angle (with improvement ranging from 82% to 95% for the four groups of patients). The least degree of improvement was noted for the labiomandibular fold (with improvement ranging from 12% to 53% for the four groups of patients). The remaining anatomic variables showed an overall improvement ranging from 61% to 85%. CONCLUSION: The suture suspension technique is a safe, reproducible technique that allows for a staged rejuvenation of the neck. As an alternative for the early rhytidectomy candidate, it produces excellent patient satisfaction outcomes with long-term corrections for the majority of patients. As compared with previously described platysmaplasty techniques, suture suspension platysmaplasty has the benefit of endurance. This article documents that the corrections can last at least 13 years with virtually unchanged aesthetic results.

Follow-Up Studies↗

An optimised protocol for plant regeneration from embryogenic suspension cultures of date palm, Phoenix dactylifera L., cv. Deglet Nour.

An improved protocol is described for the large-scale micropropagation of an elite date palm ( Phoenix dactylifera L.) cultivar, Deglet Nour. Clonal plants were regenerated from somatic embryos derived from highly proliferating suspension cultures. Friable embryogenic calli were initiated from both leaf and inflorescence explants. Suspension cultures consisting of pro-embryonic masses were established from calli showing a high competency for somatic embryogenesis. The subculture of suspensions in liquid medium enriched with low amounts of plant growth regulators (1 mg l(-1) 2,4-dichlorophenoxyacetic acid with 300 mg l(-1) charcoal) resulted in the differentiation of large numbers of somatic embryos. The productivity of the cultures increased 20-fold (from 10 to 200 embryos per month per 100 mg fresh weight of embryogenic callus) when embryogenic suspensions were used instead of standard cultures on solid media. The overall production of somatic embryos reached 10,000 units per litre per month. Partial desiccation of the mature somatic embryos, corresponding to a decrease in water content from 90% to 75%, significantly improved germination rates (from 25% to 80%). The cutting back of the cotyledonary leaf was also found to stimulate embryo germination. Flow cytometric analysis showed that the micropropagation protocol followed here did not affect the ploidy level of somatic embryo-derived plantlets.

2,4-Dichlorophenoxyacetic Acid↗

Determination of cell concentration in a plant cell suspension using a fluorescence microplate reader.

Microscopic counting of plant cells is a very tedious and time-consuming process and is therefore seldom used to evaluate plant cell number on a routine basis. This study describes a fast and simple method to evaluate cell concentration in a plant cell suspension using a fluorescence microplate reader. Eschscholtzia californica cells were fixed in a mix of methanol and acetic acid (3:1) and stained with a fluorescent DNA binding dye (Hoechst 33258). Readings were done in a fluorescence microplate reader at 360/465 nm. Specific binding of the dye to double-stranded DNA was significantly favored over unspecific binding when 1.0 M Tris buffer at pH 7.5 containing 1.0 M NaCl and 75 microg ml(-1) of Hoechst 33258 was used. Fluorescence readings must be done between 4 min and 12 min following the addition of the staining solution to the sample. The microplate counting method provides a convenient, rapid and sensitive procedure for determining the cell concentration in plant cell suspensions. The assay has a linear detection range from 0.2 x 10(6) cells to 10.0 x 10(6) cells per milliliter (actual concentration in the tested cell suspension). The time needed to perform the microplate counting was 10% of that needed for the microscopic enumeration. However, this microplate counting method can only be used on genetically stable cell lines and on asynchronous cell suspensions.

Bisbenzimidazole↗

Ciliogenesis in submersion and suspension cultures of human nasal epithelial cells.

Human nasal respiratory cells lose cilia in submerged cultures. This study compares the effect of extracellular matrix (ECM) molecules of the basal lamina on ciliogenesis in submerged cell cultures to ECM-free suspension cultures. Respiratory mucosa of nasal turbinates was the routine source for the cultures of nasal epithelial cells. For the submersion cultures, enzymatically isolated cells were seeded either on a layer of lethally irradiated ((60)Co, 60 Gy) murine 3T3-feeder fibroblasts or on an ECM-coated culture flask. For suspension cultures, the flasks were rotated for 3 days after cell seeding. In ECM-coated flasks, epithelial cell attachment and confluence was promoted and always much better than in cultures on a feeder layer. Respiratory cells lost cilia during the first 5 weeks in submerged cultures. Genesis of new, actively beating cilia was seen after 5-6 weeks when plastic culture dishes were coated with ECM molecules. Cells grown on uncoated plastic dishes together with 3T3-fibroblasts showed no ciliogenesis. Spheroids of epithelial cells in suspension cultures lost cilia during the 1st week and developed new cilia after 1-2 weeks in vitro. Our results suggest that ECM molecules are not the only signal for ciliary differentiation of respiratory cells in vitro, because suspension cultures are ECM free. However, the presence of ECM molecules in submerged cell cultures promotes the attachment and early confluence of seeded epithelial cells with a high density of cuboidal epithelial cells. The specific cellular shape and intense intercellular contact of these cuboidal cells may be among the most important signals inducing terminal differentiation and ciliogenesis.

Cells, Cultured↗

Comparison of intact and denuded amniotic membrane as a substrate for cell-suspension culture of human limbal epithelial cells.

BACKGROUND: We have previously developed a limbal epithelial culture system using a cell-suspension method on denuded amniotic membrane (AM). However, other workers reported that intact AM is advantageous for limbal epithelial culture in that it preserves stem cell characteristics. In this study, we cultivated human limbal epithelial cell-suspensions on both intact and denuded AM and compared the morphology and adhesion of the limbal epithelial cells on these two substrates. METHODS: Human limbal epithelial cells were dissociated from donor eyes using dispase and gentle pipetting and then seeded onto intact and denuded AM as cell suspension. Limbal epithelial cells on AM were co-cultured with a MMC-treated 3T3 fibroblast feeder layer and epithelial differentiation was promoted by air lifting. Cultures were examined by light, scanning and transmission electron microscopy and differences in cellular attachments and intercellular spacing were quantified. Basement membrane complexes were examined by indirect immunofluorescence. RESULTS: Limbal cells grown on denuded AM were well stratified and differentiated. Cells were well attached to each other and to the basement membrane. In contrast, limbal cells cultured on intact AM failed to stratify and in places formed a monolayer. The culture on denuded AM had significantly (P<0.001) more desmosomal junctions as well as significantly (P<0.001) more junctional attachments to the carrier than the intact culture. In addition, the intercellular spaces between cells cultivated on denuded AM were significantly (P<0.001) smaller than those between cells grown on the intact substrate. In cultures on both denuded and intact AM, the basement membrane zone displayed a positive staining for collagen VII, integrins alpha-6 and beta-4 and laminin 5. CONCLUSIONS: We successfully cultivated well-stratified and -differentiated limbal cells on denuded AM, while on the intact AM limbal cells failed to stratify and in places formed only a monolayer of cells. The limbal cells cultivated on denuded AM were well attached to the AM stroma and were morphologically superior to the limbal epithelium cultivated on intact AM. We conclude that for purposes of transplantation of differentiated epithelial sheets, denuded AM is probably the more practical carrier for human limbal epithelial cell cultures when using our cell-suspension culture system.

3T3 Cells↗

Hindlimb suspension reduces muscle regeneration.

Exposure of juvenile skeletal muscle to a weightless environment reduces growth and satellite cell mitotic activity. However, the effect of a weightless environment on the satellite cell population during muscle repair remains unknown. Muscle injury was induced in rat soleus muscles using the myotoxic snake venom, notexin. Rats were placed into hindlimb-suspended or weightbearing groups for 10 days following injury. Cellular proliferation during regeneration was evaluated using 5-bromo-2'-deoxyuridine (BrdU) immunohistochemistry and image analysis. Hindlimb suspension reduced (P < 0.05) regenerated muscle mass, regenerated myofiber diameter, uninjured muscle mass, and uninjured myofiber diameter compared to weightbearing rats. Hindlimb suspension reduced (P < 0.05) BrdU labeling in uninjured soleus muscles compared to weight-bearing muscles. However, hindlimb suspension did not abolish muscle regeneration because myofibers formed in the injured soleus muscles of hindlimb-suspended rats, and BrdU labeling was equivalent (P > 0.10) on myofiber segments isolated from the soleus muscles of hindlimb-suspended and weightbearing rats following injury. Thus, hindlimb suspension (weightlessness) does not suppress satellite cell mitotic activity in regenerating muscles before myofiber formation, but reduces growth of the newly formed myofibers.

Animals↗

Determination of optimal transport conditions for biopsies of human articular cartilage as well as for suspensions of cultured chondrocytes used in autologous transplantation.

Autologous transplantation of chondrocytes is currently being promoted as a novel approach for the treatment of deep cartilage lesions. Briefly, the method involves enzyme-mediated release of chondrocytes from cartilage biopsies, the expansion of cells by in vitro cultivation and their re-implantation into the defect. The success of this technique depends on many factors including transport conditions for both, cartilage biopsies from the operating hall to the laboratory and the return transport of final suspension of cultured chondrocytes. To determine the extent of cellular damage in biopsies, chondrocytes were enzymatically isolated following a few days of tissue preservation in different tissue culture media. The proportion of dead cells was assessed by Trypan blue staining and counting. The viability was not dependant of the type of the medium used and remained approximately 50% in all samples, even after 72 h. To develop optimal conditions for transport of final chondrocyte suspension, isolated cells were firstly grown in monolayer cultures. Cell suspensions in media with different additives were injected into special glass containers used for the transport and left at 4 degrees C or 25 degrees C for up to 3 days. During this period every 24 h the samples were taken and viability as well as apoptosis levels were assessed. Viability of cells in suspensions at 25 degrees C decreased significantly and became inadequate already after 48 h. In contrast to that, the proportion of viable cells at 4 degrees C remained above 80% even after 48 h. In the majority of the samples, culture medium containing serum and vitamin C provided the best conditions for long-term preservation of chondrocytes.

Apoptosis↗

Class-1 hemoglobins, nitrate and NO levels in anoxic maize cell-suspension cultures.

Nitric oxide (NO) is a reactive gas involved in many biological processes of animals, plants and microbes. Previous work has demonstrated that NO is formed during hypoxia in alfalfa ( Medicago sativa L.) root cultures and that the levels of NO detected are inversely related to the levels of expression of class-1 hemoglobin expressed in the tissue. The objectives of this study were: to examine whether NO is produced in transgenic maize ( Zea mays L.) cell-suspension cultures exposed to anoxic growth conditions; to determine whether a similar relationship existed between a class-1 hemoglobin and the amount of NO detected under these conditions; and, to estimate the route of formation and breakdown of NO in the tissue. Maize cell-suspension cultures, transformed to express the sense or antisense strands of barley hemoglobin were used to overexpress or underexpress class-1 hemoglobin. A maize cell-suspension culture transformed with an empty vector was used as a control. Up to 500 nmol NO (g FW)(-1) was detected in maize cells exposed to low oxygen tensions for 24 h. The steady-state levels of NO in the different cell lines under anoxic conditions had an inverse relationship to the level of hemoglobin in the cells. There was no detectable NO produced under aerobic growth conditions. Spectroscopic data demonstrated that recombinant maize hemoglobin reacted with NO to form methemoglobin and NO(3)(-). Nitrate was shown to be a precursor of NO in anoxic maize cell-suspension cultures by using (15)NO(3)(-) and electron paramagnetic resonance spectroscopy, suggesting that NO is formed via nitrate reductase during hypoxia. The results demonstrate that NO is produced in plant tissues grown under low oxygen tensions and suggest that class-1 hemoglobins have a significant function in regulating NO levels.

Cell Culture Techniques↗

Cell suspension cultures of Populus tremula x P. tremuloides exhibit a high level of cellulose synthase gene expression that coincides with increased in vitro cellulose synthase activity.

Compared to wood, cell suspension cultures provide convenient model systems to study many different cellular processes in plants. Here we have established cell suspension cultures of Populus tremula L. x P. tremuloides Michx. and characterized them by determining the enzymatic activities and/or mRNA expression levels of selected cell wall-specific proteins at the different stages of growth. While enzymes and proteins typically associated with primary cell wall synthesis and expansion were detected in the exponential growth phase of the cultures, the late stationary phase showed high expression of the secondary-cell-wall-associated cellulose synthase genes. Interestingly, detergent extracts of membranes from aging cell suspension cultures exhibited high levels of in vitro cellulose synthesis. The estimated ratio of cellulose to callose was as high as 50 : 50, as opposed to the ratio of 30 : 70 so far achieved with membrane preparations extracted from other systems. The increased cellulose synthase activity was also evidenced by higher levels of Calcofluor white binding in the cell material from the stationary-phase cultures. The ease of handling cell suspension cultures and the improved capacity for in vitro cellulose synthesis suggest that these cultures offer a new basis for studying the mechanism of cellulose biosynthesis.

Benzenesulfonates↗