Stain Uptake Research Articles

Toxinological studies of the venom from Cassiopea xamachana nematocysts isolated by flow cytometry

The tentacle epithelial tissue of Cassiopea xamachana contains nematocysts and symbiotic algal particles. These two structures were dissociated, analyzed and sorted by flow cytometry. A simple separating method was developed utilizing the algal chlorophyll autofluorescence and the nematocysts' fluorescence after the uptake of fluorescent stains. A five-fold increase in mouse lethality; significantly more potent hemolytic and cytosensing activities; as well as a cleanup in the capillary electropherogram and SDS gel profiles for the crude nematocyst venom preparations prepared by fluorescence activated cell sorting (FACS), was observed relative to alternative methods. Because the hemolytic potency of pre-sorting nematocyst venom was minimal and the post-sorting counterpart was significantly positive, the possibility that algae inhibited the venom's toxinological activity was considered.

Collagen as a model system to investigate the use of aspirin as an inhibitor of protein glycation and crosslinking

Aspirin has been shown to be a powerful inhibitor of post-Amadori Maillard reactions, although the exact mechanism of this action remains unclear. We have used corneal and scleral collagen as a model system: (i) to assess how aspirin, either alone or in combination with sugars, affects the surface charge distribution along the collagen fibrils; (ii) to see how sugars and/or aspirin affect the swelling properties of the cornea; and (iii) to see if sugars and/or aspirin change the distribution of water within the corneal stroma as the tissue swells. Charge changes were detected by examining changes in the uptake of phosphotungstate ions as seen in the electron microscope. Swelling was measured by monitoring the uptake of water as a function of swelling time, and water distribution was determined by using synchrotron X-ray diffraction to follow changes in the interfibrillar Bragg spacing as the cornea swells. Aspirin has a marked effect on the positive staining pattern of scleral collagen. This is different to the changes in stain uptake produced by glycation. Incubation with both sugars and aspirin showed that the sugar binding takes precedence over the effects of aspirin which, in turn, suggests that protein acetylation by aspirin is unlikely under these circumstances. However, aspirin completely suppresses corneal swelling. Even when the aspirin is removed, swelling in distilled water is reduced, and this is accompanied by changes in the water distribution. The results suggest that water is more evenly distributed in aspirin-treated corneas that are subsequently swollen than in swollen glycated corneas. Fructation, glucation and ribation on their own have little effect on the uptake of water as the cornea swells. This suggests that any sugar-derived crosslinks formed at this stage do not limit swelling.

Low temperature tolerance and cold acclimation for seedlings of three Mojave Desert Yucca species exposed to elevated CO2

Leaf tolerance to low temperatures, as determined by vital stain uptake and chlorophyll a fluorescence, was compared for seedlings of three Yucca species native to the south-western United States: Yucca brevifolia, which is distributed throughout the Mojave Desert;Yucca schidigera, which occurs in both coastal and desert California; and Yucca whipplei, which is primarily coastal but occurs in portions of the Mojave Desert. Seedlings maintained at day/night glasshouse air temperatures of 40/25°C or 20/5°C, and under ambient (360 μmol mol−1) or elevated (700 μmol mol−1) levels of CO2were compared to test the hypothesis that cold acclimation and freezing tolerance are enhanced by exposure to elevated CO2. Plants maintained at elevated CO2had greater low-temperature tolerance compared to controls, yet a larger shift in survival was attributable to the downward shift in day/night temperatures. Low-temperature tolerance was similar to extreme minimum air temperatures for the collection sites averaged over the period 1961 to 1990. For seedlings exposed to elevated CO2, low-temperature tolerance was −11·9°C for Yucca brevifolia, −9·6°C for Y. schidigera, and −13·5°C for Y. whipplei. Elevated CO2caused excitation energy transfer in Photosystem II (measured as FV/FM) to be maintained at lower temperatures for Yucca brevifolia and Y. whipplei. ΦPSII at low temperatures was increased due to elevated CO2for Y. brevifolia only. The results suggest that survival during episodic sub-zero temperature events will be enhanced for seedlings of these three yucca species in a future elevated CO2environment.

Induction of macrophage apoptosis by ceramic and polyethylene particles in vitro

The purpose of this study was to investigate in vitro the presence of apoptotic cell death after macrophage stimulation with different ceramic (Al 2O 3 and ZrO 2) and high density polyethylene (HDP) particles. We also analyzed the effects of particle size, concentration, and composition. The J774 mouse macrophage cell line was exposed to commercial particles of different sizes (up to 4.5 μm) and concentrations (up to 500 particles per macrophage). Fluorescence microscopy and DNA laddering were used to investigate the presence of apoptosis in cell cultures after 24 h of incubation. Fluorescence microscopy of propidium iodide stained cells showed two characteristic morphological features that occur in apoptotic cells, namely nuclear condensation and heterogeneity of stain uptake. The effect of ceramic particles on apoptotic nuclear morphology was size- and concentration-dependent and reached a plateau above 150 particles per macrophage at 1.3 μm. With regards to composition, we did not find any difference in cell morphology between Al 2O 3 and ZrO 2. Ceramic and HDP particles induced DNA fragmentation into oligonucleosomes as evidenced by DNA laddering, another characteristic of apoptosis. The induction of DNA laddering was size- and concentration-dependent whereas particle composition (Al 2O 3 vs. ZrO 2 and Al 2O 3 vs. HDP) had no effect. In conclusion, our results demonstrated that ceramic and HDP particles induce macrophage apoptotic cell death in vitro and open doors for possible modulation of debris-induced periprosthetic osteolysis.

Immunohistochemical localization of nephrocalcin, a kidney-specific glycoprotein, to renal cell carcinoma

Objectives. Nephrocalcin (NC), an acidic glycoprotein produced by renal proximal tubule cells and functioning as an inhibitor of calcium oxalate monohydrate crystalization, has been previously shown to have increased urinary excretion in patients with renal cell carcinoma (RCC). The current study uses immunohistochemical techniques to localize NC to cells of primary RCC. Methods. We studied 29 kidneys removed because of RCC. Slides were deparaffinized and stained after incubating with anti-NC antibody by using the avidin-biotin-peroxidase complex techniques. Uptake of stain by tumor cells and adjacent normal renal cells was compared. Results. Twenty-seven kidneys (93%) showed positive staining for RCC tumor cells; 2 kidneys staining positive for normal proximal tubule cells failed to stain adjacent RCC tumor cells (7%). Conclusions. The data suggest that enhanced production of urinary NC in patients with RCC derives from cells of the primary tumor.

Glycation changes the charge distribution of type I collagen fibrils.

In aging and diabetes, glycation of collagen molecules leads to the formation of cross-links that could alter the surface charge on collagen fibrils, and hence affect the properties and correct functioning of a number of tissues. The electron-optical stain phosphotungstic acid (PTA) binds to positively charged amino acid side-chains and leads to the characteristic banding pattern of collagen seen in the electron microscope; any change in the charge on these side-chains brought about by glycation will affect the uptake of PTA. We found that, upon glycation, a decrease in stain uptake was observed at up to five regions along the collagen D-period; the greatest decrease in stain uptake was apparent at the c1 band. This reduction in PTA uptake indicates that the binding of fructose leads to an alteration in the surface charge at several sites along the D-period. Not all lysine and arginine residues are involved; there appear to be specific residues that suffer a loss of positive charge.

Vascular Endothelial Cells Generate Peroxynitrite in Response to Carbon Monoxide Exposure

Carbon monoxide causes a perivascular oxidative injury in animals, and we tested the hypothesis that endothelial cells could be a source of the injurious oxidants. Studies were undertaken to assess whether exposure to carbon monoxide would cause cultured bovine pulmonary artery endothelial cells to liberate reactive species. Concentrations of carbon monoxide between 11 and 110 nM caused progressively higher concentrations of nitric oxide to be released by endothelial cells based on measurements of nitrite and nitrate. Intracellular production of peroxynitrite was indicated by elevated concentrations of nitrotyrosine, and extracellular liberation of peroxynitrite was indicated by oxidation of p-hydroxyphenylacetic acid and dihydrorhodamine-123. Carbon monoxide did not disturb mitochondrial function based on the rate of oxygen consumption, intracellular production of hydrogen peroxide, and the ability of cells to reduce 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. Carbon monoxide also did not alter arginine transport by cells or nitric oxide synthase activity, but it was found to increase steady state levels of nitric oxide by competing for intracellular binding sites. Acute cytotoxicity from carbon monoxide, assessed as radioactive chromium leakage, was due to nitric oxide-derived oxidants. A delayed cell death, whose mechanism is not entirely clear, was also demonstrated by chromium leakage and uptake of vital stain. These findings offer a possible mechanism for adverse health effects caused by carbon monoxide at concentrations ranging from the relatively low levels in polluted environments to levels typically encountered with life-threatening poisoning. Carbon monoxide causes oxidative stress by a novel mechanism involving a competition for intracellular binding sites which increases steady state levels of nitric oxide and allows for generation of peroxynitrite by endothelium.

Isolation of porcine pancreatic islets for xenotransplantation studies: Effects of low collagenase digestion temperatures

Abstract: Islets of Langerhans were isolated from porcine pancreata by a modification of our previously described method. The modification involved the use of a low temperature of collagenase digestion (30°C) during the process of islet isolation. The resulting islets were then evaluated in vitro and in vivo and compared to islets isolated at the regular 37°C temperature.The islets produced at the low temperature were more compact compared to the control islets. In the dextran density gradient these islets were deposited at the interface of the 1.060 and 1.068 g/ml density bands as compared to 1.050 and 1.060 g/ml for the control islets. In addition, the experimental islets contained a higher proportion of compact, unfragmented islets (68%) compared to the regular islets (55%), and their uptake of the dithizone stain was considerably slower than with the control islets. All ten batches of freshly isolated microencapsulated islets produced at both temperatures responded to the glucose stimulation. After 4 weeks of in vitro culture the islets of both groups microencapsulated in alginate‐polylysine‐alginate (APA) microcapsules still retained glucose responsiveness, with the experimental islets demonstrating significantly higher responsiveness to the high glucose (16.7 mM) and 0.1 mM IB MX stimulation. The morphology of unencapsulated islets in the experimental group following 4 weeks of in vitro culture indicates much firmer islet structure compared to the control islets. In addition, the unencapsulated experimental islets following the 4 week culture were still found to have secreted insulin when exposed to glucose. In transplantation studies both the experimental and the control islets normalized diabetic hyperglycemia in diabetic mice in a comparable fashion. In general, the low temperature digestion results in superior islets in terms of their morphology, viability, and physiological function.

Effects of fluoride on structure and function of canine gastric mucosa.

These studies were done to determine the effects of fluoride (F) on the structure and function of the canine gastric mucosa and the possible protective effects of 16,16-dimethyl-prostaglandin E2 (dmPGE2). A portion of the stomach with its vascular supply intact was mounted in a two-compartment chamber, one side of which contained a control solution. Minor effects were caused by exposure to 1 mmol/liter F. Both 5 and 10 mmol/liter F caused marked increases in the fluxes of water and Na, K, and H ions; mucus secretion; and tissue swelling and redness. The extent of these changes did not increase appreciably upon exposure to 50 or 100 mmol/liter F. Histological findings included marked thinning of the surface cell layer, reduced uptake of PAS stain, localized exfoliation and necrosis of surface cells, acute gastritis, and edema. It was concluded that: (1) the threshold F concentration for effects on the structure and function of the gastric mucosa was approximately 1 mmol/liter; (2) the maximum or near-maximum effects were caused by 10 mmol/liter F; (3) the effects persisted for at least 6 hr after the exposure; and (4) dmPGE2 (0.5 microg/ml) did not attenuate the effects induced by F.

RAPID PREDICTION OF ALUMINUM TOLERANCE IN ALFALFA

Alfalfa (Medicago sativa L.), one of the most important forage legumes in the United States, has been recognized as an aluminum-sensitive species (Kemp-Glass et al., 1993). Hematoxylin staining has been used to evaluate differences in root growth and stain uptake between sensitive and resistant individuals in wheat (Ruiz-Torres et al., 1992). Attention in this study is focused on the hematoxylin staining pattern because the procedure is simple and rapid. Ten alfalfa cultivars were used: `Apollo', `ARC', `Foundation Vemal', `Shenandoah', `Spreador 2', `WL 311', `Saranac', `Saranac AR', `Cimarron', and `Cimarron VR'. Twenty seeds of each were stained in a solution of hematoxylin for 2 days. After staining, the seedlings were transferred to a potting medium for 14 days. After 14 days, plantlets were transferred to Porters soil (pH 4.5, 80% aluminum saturation) and grown in the greenhouse for 60 days. After 60 days, fresh and dry root and shoot weights were taken. Root length densities were determined and these parameters were compared to the tolerance level predicted by hematoxylin stain. Results of stain correlate with biomass at highly significant levels and will be of great use in the development of an acid/aluminum-tolerant alfalfa.

Viability Assessment of Turkey Sperm Using Fluorescent Staining and Flow Cytometry

Turkey sperm viability was evaluated using several fluorescent stains both singularly and in combination. Dilution curves and several extenders were used to determine optimal stain concentrations. Semen was collected from eight toms, pooled, diluted, stained, and evaluated microscopically within 2 h of collection. Replicates were assessed for both viable and nonviable sperm (green and red fluorescence, respectively) using flow cytometry. SYBR-14, which likely requires membrane potential for optimal fluorescence, or Calcein AM (CAL), which assesses the membrane integrity of cells (both green fluorescence), in combination with propidium iodide (PI) to stain the dead or degenerating cells (red fluorescence) provided optimal results. Sperm were killed by unprotected freeze-thawing to provide mixed aliquots containing known amounts of fresh:killed sperm. The percentage of viable sperm, as determined by SYBR-14 with PI or CAL with PI staining, were 76.6, 58.8, 39.3, 20.1, .8, and 73.5, 55.8, 36.0, 17.1, .4, respectively, for ratios of 100:0,75:25, 50:50, 25:75, and 0:100 of fresh:killed mixtures. Semen from 30 individual toms was collected on 2 d and examined in replicate using these staining combinations. The proportion of viable sperm, as indicated by uptake of SYBR-14 or CAL stain, ranged from 55.8 to 86.7 and 38.0 to 86.1, respectively. Staining combinations were effective in estimating the viability of turkey sperm and could be useful for monitoring sperm viability before and after storage.

Flow cytometric DNA content analysis of fibropapillomas in green turtles Chelonia mydas

DAO Diseases of Aquatic Organisms Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsSpecials DAO 22:13-18 (1995) - doi:10.3354/dao022013 Flow cytometric DNA content analysis of fibropapillomas in green turtles Chelonia mydas Papadi GP, Balazs GH, Jacobson ER In an attempt to characterize biologic properties of green turtle fibropapillomatosis (GTFP), blood cells, dermis of normal skin, dermis of cutaneous fibropapillomas, and visceral fibroblastic nodules of green turtles Chelonia mydas with cutaneous fibropapilloma were examined by flow cytometry. DNA of each sample was stained with propidium iodide and histograms were recorded utilizing a FACSCAN flow cytometer. All samples examined had similar histograms, indicative of normal cell cycles and diploid profiles. Small S phase and G2M fractions (<5%) indicated a low mitotic activity and a nonproliferating state. Statistical analyses were performed on the coefficient of variation (CV) of the G1 peak for the various tissues analyzed. The mean CV for blood from all the unaffected turtles was 3.21 while that from turtles afflicted with GTFP was 3.43. CVs of normal skin from healthy turtles and turtles afflicted with GTFP were 4.25 and 4.41 respectively. The mean CV for fibropapillomas was 5.27, a number which, though higher than either blood or normal skin, probably reflects the difference in stain uptake characteristics between fibropapillomas and the other 2 tissues rather than any inherent DNA or ploidy difference. Fibropapilloma . Flow cytometry . Green turtle . Chelonia mydas Full text in pdf format PreviousNextExport citation RSS - Facebook - Tweet - linkedIn Cited by Published in DAO Vol. 22, No. 1. Publication date: May 04, 1995 Print ISSN:0177-5103; Online ISSN:1616-1580 Copyright © 1995 Inter-Research.

Exogenous Abscisic Acid Mimics Cold Acclimation for Cacti Differing in Freezing Tolerance.

The responses to low temperature were determined for two species of cacti sensitive to freezing, Ferocactus viridescens and Opuntia ficus-indica, and a cold hardy species, Opuntia fragilis. Fourteen days after shifting the plants from day/night air temperatures of 30/20[deg]C to 10/0[deg]C, the chlorenchyma water content decreased only for O. fragilis. This temperature shift caused the freezing tolerance (measured by vital stain uptake) of chlorenchyma cells to be enhanced only by about 2.0[deg]C for F. viridescens and O. ficus-indica but by 14.6[deg]C for O. fragilis. Also, maintenance of high water content by injection of water into plants at 10/0[deg]C reversed the acclimation. The endogenous abscisic acid (ABA) concentration was below 0.4 pmol g-1 fresh weight at 30/20[deg]C, but after 14 d at 10/0[deg]C it increased to 84 pmol g-1 fresh weight for O. ficus-indica and to 49 pmol g-1 fresh weight for O. fragilis. Four days after plants were sprayed with 7.5 x 10-5 M ABA at 30/20[deg]C, freezing tolerance was enhanced by 0.5[deg]C for F. viridescens, 4.1[deg]C for O. ficus-indica, and 23.4[deg]C for O. fragilis. Moreover, the time course for the change in freezing tolerance over 14 d was similar for plants shifted to low temperatures as for plants treated with exogenous ABA at moderate temperatures. Decreases in plant water content and increases in ABA concentration may be important for low-temperature acclimation by cacti, especially O. fragilis, which is widely distributed in Canada and the United States.

Natural history of intraepithelial neoplasia in humans with implications for cancer chemoprevention strategy : CW Boone, GJ Kelloff, VE Steele. Cancer Res 52:1651–1659, 1992

Intraepithelial neoplasia is of critical importance to the cancer chemoprevention field because it is a target condition for which drugs must be sought that will prevent its development or stop its progression. The term "dysplasia" refers to the morphological alterations that characterize intraepithelial neoplasia and according to many authors consists of seven basic morphological changes that occur in the majority of human epithelia, as well as in the epithelium of mouse skin papillomas induced by 7,12-dimethylbenz(a)anthracene and 12-O-tetradecanoylphorbol-13-acetate: increased nuclear size; altered nuclear shape; increased nuclear stain uptake; nuclear pleomorphism (increased variation in nuclear size, shape, and stain uptake); increased mitoses; abnormal mitoses; and disordered or absent maturation. Clonal evolution appears to begin early in the neoplastic process during intraepithelial neoplasia. Aneuploidy has been found during intraepithelial neoplasia in many human epithelia, and, in association with other forms of genetic instability, may provide the increase in genetically variant cells required for clonal evolution to occur. It is postulated that two major factors affecting the rate of progression of intraepithelial neoplasia are the cellular mutation rate, which is enhanced by environmental carcinogens, and the cellular proliferation rate, which is enhanced by agents that include sex hormones, inducers of chronic inflammation, and irritant chemicals which stimulate reactive hyperproliferation. A preferred chemoprevention strategy should consist of the development of drugs and drug combinations which will block mutagenic carcinogens or prevent epithelial hyperproliferation or its causes. Two examples of the induction of regression of intraepithelial neoplasia by chemopreventive drugs are the regression of oral leukoplakia produced by beta-carotene and the regression of colorectal polyps in patients with familial polyposis produced by sulindac. It is evident that there is a strong need for more research on the induction of regression of intraepithelial neoplasia with chemopreventive agents. There is also a critical need to identify and develop biomarkers that correlate with the appearance and regression of intraepithelial neoplasia.

The natural history of intraepithelial neoplasia: relevance to the search for intermediate endpoint biomarkers.

The development of carcinomas, defined as invasive epithelial neoplasms, is preceded by a preinvasive stage termed intraepithelial neoplasia that typically lasts for years. Intraepithelial neoplasia is the target tissue for the action of chemopreventive agents and the site where biomarkers frequently develop. The term "dysplasia" refers to the morphological alterations that characterize intraepithelial neoplasia and, according to many authors, consists of seven basic changes that are the same for the majority of epithelia. These are increased nuclear size, abnormal nuclear shape, increased nuclear stain uptake, nuclear pleomorphism (increased variation in size, shape, and stain uptake), increased mitoses, abnormal mitoses, and disordered or absent differentiation. Clonal evolution appears to begin early in the neoplastic process during intraepithelial neoplasia. The use of intraepithelial neoplasia as an intermediate endpoint biomarker requires that effective chemopreventive agents cause it to regress. Two examples are the regression of dysplastic oral leukoplakia produced by beta-carotene and the regression of colonic polyps in familial polyposis patients following treatment with the nonsteroidal antiinflammatory drug sulindac. There is a critical need to identify and develop biomarkers that correlate with the appearance and regression of intraepithelial neoplasia.

A study of staining for electron microscopy using collagen as a model system—IX. The effect of tannic acid fixation

Electron optical examination of reconstituted collagen fibrils exposed to tannic acid reveals marked changes in staining behaviour. Positive staining in solutions of tungstate salts, where uptake of the heavy metal anions occurs on the positively charged side-chains of arginyl, lysyl and hydroxylysyl residues, reveals some reduction in stain uptake following tannic acid treatment. This reduced uptake is consistent with inhibition of staining at residues known, from independent biochemical evidence, to interact with tannic acid. Negative staining shows that tannic acid introduces some additional bulk into the fibril structure, although this is not as great as that induced by glutaraldehyde fixation. Staining patterns from doubly fixed specimens, together with thermal stability measurements on collagen gels, show that tannic acid fixation does not preclude a subsequent reaction with glutaraldehyde.

Cell proliferation and oxidative stress: basis for anticancer drugs

Synopsis:A wide variety of normal and malignant mammalian cells, as well as inflammatory cells, release extracellular active oxygen species such as superoxide and hydrogen peroxide. Both these species at low levels can stimulate growth and growth responses in a range of normal and malignant cell types. In order to assess the significance of such findings, superoxide dismutase and catalase were added exogenously to the culture medium of hamster (BHK-21) and rat (208F) fibroblasts (non-transformed or oncogene transformed). This resulted in a reduction of cell proliferation and increased cellular uptake of trypan blue stain, suggesting that superoxide and/or hydrogen peroxide may have important roles as extracellular ‘messengers’ promoting cell proliferation and maintaining cell viability. Superoxide dismutase mimics, like superoxide dismutase itself, are also cytostatic towards these cells and provide a basis for the development of tumour-specific antiproliferative drugs.

An Alternate Method for Staining Plastic Embedded Autoradiographs

AbstractWe have developed a rapid and reliable method for staining plastic embedded autoradiographs for in situ labeling protocols that eliminates several problems sometimes encountered with other methods, such as displacement of silver grains and uptake of stain by the emulsion layer itself. The procedure features the brief (less than one minute) application of toluidine blue to plastic embedded autoradiographs with moderate warming. The quality of staining obtained by this method is both consistent and flexible, allowing varying degrees of intensification without the danger of damaging the emulsion layer. The principle advantages of this procedure over other techniques to stain plastic embedded autoradiographs are its simplicity, rapidity and reproducibility. (The J Histotechnol 14:43, 1991)

Timing of molecular events following elicitor treatment of plant cells

Extracellular products with elicitor activity from the bean pathogen Colletotrichum lindemuthianum induced a multiplicity of changes in bean suspension cells. An immediate increase in luminol-mediated chemiluminescence which peaked within 30 min indicated production of activated oxygen species. Shut down of synthesis of discrete proteins and enhanced synthesis of other proteins was apparent by 3 h. These differences in protein profiles between elicitor-treated and control cells were more pronounced by 6 h. Other changes at 4–6 h involved perturbations in membrane functions including lipid peroxidation, altered proton efflux and uptake of a vital stain, fluorescein diacetate as well as reduced activity of the extracellular enzymes peroxidase and β glucosidase. These changes coincide with an increase in electron paramagnetic resonance signal consistent with free radical accumulation. The accumulation of phytoalexins at 6–8 h after treatment suggests that phytoalexins do not account for either free radicals detected earlier by the electron paramagnetic resonance signals or altered plant membrane function. Our data suggest that altered protein expression may be triggered by free radicals connected to activated oxygen metabolism.

Low-temperature tolerance and CO2 uptake for platyopuntias—a laboratory assessment

Fourteen accessions of Opuntia ficus-indica, O. fusicaulis, O. hyptiacantha, O. megacantha, O. rastrera, O. stricta, and O. streptacantha, whose low-temperature damage in the field in south Texas had previously been determined, were examined for low-temperature tolerance in the laboratory. Those platyopuntia accessions exhibiting the least low-temperature damage in the field had the greatest low-temperature tolerance in the laboratory, assayed using uptake of a vital stain (neutral red) by the chlorenchyma cells. All 14 accessions exhibited low-temperature acclimation as the day/night air temperatures for growth were reduced from 35°C/25°C to 25°C/15°C to 15°C/5°C, the greatest low-temperature tolerance being associated with the greatest acclimation ability. To screen for possible differences in CO2 uptake ability among the six accessions exhibiting the greatest low-temperature tolerance, the nocturnal increases in tissue acidity and osmotic pressure were determined in the laboratory for these Crassulacean acid metabolism plants. Both measures of CO2 uptake were maximal at the intermediate growth temperature, 25°C/15°C. Moreover, the two variables were highly correlated (r2 = 0·87, p