Further studies on preservation of the beating rhythm of myocardial cells in culture

1. 1. Single myocardial cells from fetal mouse heart beat spontaneously in monolayer culture. In standard medium they maintained a constant beating rate for at least 5 h. After the beating rate of individual cells had been accelerated for a short time by electrical stimulation, the original beating rate could be immediately restored by interrupting the stimulation. Quiescent myocardial cells from neonatal mouse atrium could be induced to beat by electrical stimulation and most of them ceased to beat again immediately by interrupting the stimulation.

2. 2. After the spontaneous beating of individual myocardial cells had been stopped or slowed down for a short time by incubation in medium of low temperature or high potassium or low calcium concentration, the original beating rate could be restored by replacing the cells in the original, normal medium.

3. 3. After the spontaneous beating of individual myocardial cells had been stopped by adding a metabolic inhibitor, such as 2,4-dinitrophenol or 2-deoxyglucose, the original beating rate could be restored by replacing the cells in the original, normal medium.

4. 4. Both single myocardial cells and cell clusters showed arrhythmia, including flutter and fibrillation, in medium of low potassium or high calcium concentration. After a short period of arrhythmia, the original beating rate could be restored by replacing the cells in the original, normal medium. The arrhythmia of cell clusters produced in either low potassium or high calcium medium was also corrected immediately by addition of quinidine sulfate.

     

Recombinant expression, purification, and characterization of XorKII: a restriction endonuclease from Xanthomonas oryzae pv. oryzae

An endonuclease from Xanthomonas oryzae pathovar oryzae (Xoo) KACC10331, XorKII, was recombinantly produced in Escherichia coli by applying the stationary state induction method, which was necessary to prevent the unwanted lysis of E. coli cells. XorKII was purified by immobilized metal affinity chromatography on an FPLC system. The yield was 3.5 mg of XorKII per liter of LB medium. The purified recombinant XorKII showed that it recognized and cleaved to the same site as PstI. It behaved as a dimer as evidenced by the size exclusion chromatography. The specific activity of the purified XorKII was determined to be 31,300 U/mg. The enzyme activity was monitored by cleaving lambda DNA or YEp24 plasmid as substrates. The enzyme was the most active at 10 mM Tris–HCl pH 7.0, 10 mM MgCl₂, 1 mM dithiothreitol at 37 °C. XorKII was easily inactivated by heating at 65 °C for 5 min, but retained most of the original activity after incubation at 37 °C for 24 h.     

Formation of extracellular and intracellular ice during warming of vitrified mouse morulae and its effect on embryo survival

In vitrified solutions, ice can form during warming if the concentration of the cryoprotectant is insufficient. For the cryopreservation of cells, ice is innocuous when it remains outside the cell, but intracellular ice (ICI) is lethal. We tried to estimate the conditions in which ICI forms in vitrified mouse morulae during warming. The solutions for the experiments (EFS10–EFS50) contained 10–50% ethylene glycol plus Ficoll plus sucrose. When vitrified EFS20, EFS30, and EFS40 were kept at −80 °C, they remained transparent after 3 min, but turned opaque after 60 min (EFS20, EFS30) or 24 h (EFS40). Morulae were vitrified with EFS solutions after exposure for 30–120 s at 25 °C. They were warmed by various methods and survival was assessed in culture. After rapid warming (control), survival was high with EFS30 (79–93%) and EFS40 (96–99%). After slow warming, survival decreased with both EFS30 (48–62%) and EFS40 (44–64%). This must be from the formation of ICI. To examine the temperature at which ICI formed during slow warming, vitrified embryos were kept at various sub-zero temperatures during warming. Survival with EFS30 and EFS40 decreased on keeping samples for 3 min at −80 (25–75%), −60 (7–49%), −40 (0–41%), or −20 °C (26–60%). When samples were kept at −80 °C for 24 h, the survival decreased to 0–14%. These results suggest that ICI forms at a wide range of temperatures including −80 and −20 °C, more likely between −60 and −40 °C, and the ice forms not only quickly but also slowly.     

The dominance of warming rate over cooling rate in the survival of mouse oocytes subjected to a vitrification procedure

The formation of more than trace amounts of ice in cells is lethal. The two contrasting routes to avoiding it are slow equilibrium freezing and vitrification. The cryopreservation of mammalian oocytes by either method continues to be difficult, but there seems a slowly emerging consensus that vitrification procedures are somewhat better for mouse and human oocytes. The approach in these latter procedures is to load cells with high concentrations of glass-inducing solutes and cool them at rates high enough to induce the glassy state. Several devices have been developed to achieve very high cooling rates. Our study has been concerned with the relative influences of warming rate and cooling rate on the survival of mouse oocytes subjected to a vitrification procedure. Oocytes suspended in an ethylene glycol–acetamide–Ficoll–sucrose solution were cooled to −196 °C at rates ranging from 37 to 1827 °C/min between 20 and −120 °C, and for each cooling rate, warmed at rates ranging from 139 to 2950 °C/min between −70 and −35 °C. The results are unambiguous. If the samples were warmed at the highest rate, survivals were >80% over cooling rates of 187–1827 °C/min. If the samples were warmed at the lowest rate, survivals were near 0% regardless of the cooling rate. We interpret the lethality of slow warming to be a consequence of it allowing time for the growth of small intracellular ice crystals by recrystallization.     

Adaptation to different growth temperatures modifies some mammalian cell survival responses

Chinese hamster (HA-1) cells that have been grown at 37 °C since explant several years ago can adapt themselves to grow at temperatures ranging from 32 to 41 °C. This growth adaptation is accompanied by major phenotypic changes in, for exampie, the cellular responses to 43 and 45 °C heat challenges and to ethanol challenges (0–10% in concentration). Cells grown at 39.5 °C are seen to acquire substantial heat resistance when compared with cells grown at 37 °C; resistance is even more pronounced if the growth temperature is at 41 °C. On the other hand, cells grown at 32 °C become more sensitive to heat than controls. Our results also indicate an increased resistance to ethanol of the 41 °C grown cells. By contrast the cells' X-ray survival response is affected only minimally. The changes seen are phenotypic; upon being returned to 37 °C, HA-1 cells within 34 h regain their ‘normal’ heat responses.     

A cytotoxic, photolabile cross-linking derivative of ricin : Action on various cells and application to the study of ricin toxicity

Ricin has been coupled to the cleavable, photolabile, hetero-bifunctional cross-linking reagent N-[4-(p-azidophenylazo)benzoyl] 3-aminopropyl-N¹-oxysuccinimide ester. Approx. 1.3–1.9 moles/mole of ricin are present in the conjugate. The conjugate is fully toxic when tested in the dark on intact cells. Cells, incubated with low amounts of the conjugate at 2 °C for 45 min and shifted to 37 °C for 3 h, become markedly protected against the effect of low concentrations of the toxin if irradiated after 2.5–3 h, when the toxic effect on protein synthesis in non-irradiated cells is maximal. Irradiation at intermediate times produces no or only partial protection. Less protection is obtained with high concentrations of the conjugate or in the presence of methylamine, a reagent that enhances uptake of ricin into cells.Subcellular fractionation of cells exposed in the dark to the photolabile ricin derivative for various times reveals that the conjugate is cross-linked to plasma membrane-derived fractions at early times. After 45 min at 37 °C the ricin is associated with subcellular fractions enriched in lysosomes and Golgi-derived elements and after prolonged times at 37 °C with subcellular fractions supporting RNA synthesis.     

Phosphatidylcholine mobility in liver microsomal membranes

Purified phosphatidylcholine exchange protein from bovine liver was used to exchange rat liver microsomal phosphatidylcholine for egg phosphatidylcholine. It was found that at 25 and 37°C rat liver microsomal phosphatidylcholine was completely and rapidly available for replacement by egg phosphatidylcholine. In contrast, phosphatidylcholine in vesicles prepared from total microsomal lipids could only be exchanged for about 60%. At 8 and 0°C complex exchange kinetics were observed for phosphatidylcholine in rat liver microsomes. The exchange process had neither effect on the permeability of the microsomal membrane to mannose 6-phosphate, nor on the permeability of the phosphatidylcholine vesicles to neodymium (III) cations.Purified phospholipase A₂ from Naja naja could hydrolyze some 55–60% of microsomal phosphatidylcholine at 0°C, but 70–80% at 37°C. Microsomal phosphatidylcholine, remaining after phospholipase treatment at 37°C, could be exchanged for egg phosphatidylcholine at 37°C, but at a slower rate than with intact microsomes. Microsomal phosphatidylcholine remaining after phospholipase treatment at 0 and 37°C had a lower content of arachidonic acid than the original phosphatidylcholine.These results are discussed with respect to the localization and transmembrane movement of phosphatidylcholine in liver microsomes.     

Effect of chilling on porcine germinal vesicle stage oocytes at the subcellular level

The potential subcellular consequence of chilling on porcine germinal vesicle (GV) stage oocytes was examined. Prior to in vitro maturation (IVM), Cumulus-oocyte complexes (COCs) freshly collected from antral follicles (3–6 mm in diameter) were evenly divided into four groups and immediately incubated in PVA-TL-HEPES medium at the temperature of 39 °C (control group), 23 °C (room temperature), 15 °C and 10 °C for 10 min, respectively. Following 42 h of IVM at 39 °C, the survival rates were examined. There was no significant difference between the survival rate of 23 °C chilled group and control group (77.92 and 91.89%), but the survival rate of 15 and 10 °C chilled group were significantly decreased (46.34 and 4.81%, P < 0.01). A further experiment on15 °C group showed that most oocytes died from 2 to 4 h of IVM. In order to investigate the effects of chilling on oocytes at the subcellular level, the control and 15 °C chilled group COCs fixed at different time points of the IVM cultures (2, 2.5, 3, 3.5 and 4 h of IVM) were prepared for transmission electron microscope (TEM) observation. As the result, compared with the control group, there were two significant changes in the ultrastructural morphology of 15 °C treatment group: (1) dramatic reduction of heterogeneous lipid, (2) disorganized mitochondria–endoplasmic reticulum–lipid vesicles (M–E–L) combination. These results indicate that 15 °C is a critical chilling temperature for porcine GV stage oocyte and the alteration of cellular chemical composition and the destruction of M–E–L combination maybe responsible for chilling injury of porcine oocyte at this stage.     

Effects of temperature on development of vedalia beetle, Rodolia cardinalis (Mulsant)

The effect of temperature on the development of the vedalia beetle, Rodolia cardinalis (Mulsant) (Coleoptera: Coccinellidae), fed Icerya purchasi Maskell (Homoptera: Margarodidae) under controlled laboratory conditions was studied. Adults exposed to temperatures of 25, 28, 31, 34, and 37 °C for 72 h showed 95–100% survival, however egg production was significantly reduced at 34 and 37 °C. In addition, eggs maintained at 34 °C showed reduced hatch and survival of larvae, and eggs held at 37 °C failed to hatch. The duration of each developmental stage and survival of each stage were measured at 10, 14, 18, 22, and 25 °C. There was no egg eclosion at 10 °C. The developmental time from egg to adult emergence decreased from 79 to 18 days for temperatures from 14 to 25 °C. The sex ratio was unaffected by these temperatures. The lower developmental temperature threshold of R. cardinalis was estimated to be 10.8 °C and the degree–day accumulation was calculated as 279 for development from egg to adult eclosion. These results will guide further research designed to optimize management of vedalia populations in the San Joaquin Valley of California.     

Heat-sensitive development of the phagocytotic organelle in a Tetrahymena mutant

A laboratory-induced mutant with heat-sensitive development of the phagocytotic organelle has been isolated in Tetrahymena pyriformis, syngen 1; the mutant cells form food vacuoles at 30 °C, but not after incubation at 37 °C. Mutant cells transferred to 37 °C undergo a maximum of 3–5 doublings, but a sizeable fraction remains viable for several days. Results of temperature shift-up experiments reveal that an oral apparatus (OA) constructed at 30 °C remains functional at 37 °C, while one constructed at 37 °C is non-functional with regard to phagocytosis. Preliminary cytological observations reveal severe structural abnormalities of the OA. Thus the mutant appears to be primarily affected in the morphogenesis of the OA. The phenotypic effect of the mutation is reversible by a temperature shiftdown. Changes in phenotype caused by temperature shifts in either direction can occur even in stationary or starved cultures. Cell division is not required for the resumption of phagocytosis after a temperature shiftdown. Null-formers obtained at the first doubling after a temperature shift-up can divide at least once more, indicating that a functional OA is not required for cell division at any stage of the cell cycle. Mutants defective in phagocytosis may prove useful in gaining deeper understanding of this mechanism and its relationship to other cellular processes.     

Down-regulation of Transforming Growth Factor-β Receptors: Cooperativity between the Types I, II, and III Receptors and Modulation at the Cell Surface

The types I, II, and III receptors (RI, RII, RIII) for transforming growth factor-β (TGF-β) become down-regulated in response to ligand, presumably via their internalization from the cell surface. This report examines the down-regulation of full-length RI, RII, and RIII in cells endogenously or transiently expressing these receptors. Down-regulation occurred rapidly (within 2 h after TGF-β1 treatment at 37°C) and showed a dose response, between 10 and 200 pM TGF-β1, in cells expressing RI, RII, and RIII (Mv1lu and A549 cells). A comparison between Mv1Lu and mutant cell derivatives R-1B (lacking RI) or DR-26 (lacking RII) indicated that all three receptors were necessary for efficient down-regulation. Down-regulation experiments, utilizing TGF-β-treated 293 cells transiently expressing different combinations of these receptors indicated that neither RII or RIII were down-regulated when expressed alone and that RI was required for maximal down-regulation of RII. RII and RIII were partially down-regulated when these receptors were coexpressed in the absence of RI (in R-1B and 293 cells). Surprisingly, TGF-β receptors were partially down-regulated in Mv1Lu, A549, and 293 cells treated with TGF-β1 at 4°C. Microscopic examination of 293 cells coexpressing RI fused to green fluorescent protein (RI–GFP) and RII indicated that, after treatment with TGF-β1 at 4°C, RI–GFP formed aggregates at the cell surface at this temperature. RI–GFP was not detected at the surface of these cells after TGF-β1 treatment at 37°C. Our results suggest a two phase mechanism for TGF-β1 receptor down-regulation involving receptor modulation (aggregation) at the cell surface and internalization.     

Temperature compensation in the mammalian cell cycle

Random and synchronous V79 cells were shifted from 37.5 °C to temperatures between 29 ° and 41 °C. Intermitotic time determinations of random cultures showed an increase in generation time and a broadening in the distribution of generation times in cells whose cycle spanned the temperature shift, but only a slight increase in generation time after one generation at temperatures between 34 °–40 °C. At 33.5 °C and below there was a stepwise increase in generation time. When cells grown at non-standard temperatures were allowed to habituate for 48 h at the altered temperature prior to analysis, the increase in median intermitotic time was slightly less in comparison to analyses done after only one generation following the temperature step. The Q₁₀ for cell division of cells growing at temperatures from 34 ° to 40 °C was between 1.15 and 1.26, suggesting that the mammalian cell cycle is temperature compensated over a limited (6–7 °C) temperature span. Mammalian cells in culture appear to have the same capacity for temperature compensation in their cell cycle as do unicellular eukaryotes. The fact that cycle time at lower temperatures increases in a discrete manner is taken as evidence for a quantal clock.     

Chronic exposure to inhaled anesthetics increases cholesterol content in Acholeplasma laidlawii

Acholeplasma laidlawii cells were grown in cholesterol-enriched medium and exposed continuously to either air (control), 4.0 vol.% halothane in air at 1 atm pressure (4% atm halothane), or 80% cyclopropane in oxygen for 24 h at 37°C. Cells grown in the presence of 4% atm halothane or 80% cyclopropane had approximately twice as much membrane cholesterol content/mg protein as the control cells. Cells grown in an anesthetic environment also tended to have a higher membrane cholesterol/phospholipid molar ratio compared to control cells. Membranes isolated from halothane-exposed cells grown in a cholesterol-enriched medium were more ordered at 37°C (measurements were made with no anesthetic present) than membranes from control cells grown in an identically enriched medium. This difference in membrane physical state between control and anesthetic-exposed cells decreased as the temperature decreased, and disappeared at approx. 23°C. Continuous exposure of A. laidlawii to 4% atm halothane or 80% cyclopropane for 24 h did not markedly affect membrane fatty acid composition, either in cells grown on an unsupplemented medium or in cells grown in a medium enriched in myristic, palmitic or stearic acids. These results further support the hypothesis that an increased membrane cholesterol content may play a role in the tolerance or dependence that develops after chronic exposure to anesthetic agents.     

Interaction of murine colony-stimulating factor (CSF-1) with alveolar mononuclear phagocytes

Colony-stimulating factor (CSF-1) was purified from serum-free L-cell-conditioned medium (LCM) and iodinated so that we could study its interaction with murine alveolar macrophages. At 0 °C, the binding of ¹²⁵ICSF-1 to alveolar macrophages reached a stable maximum within 16 h. Under this condition, the binding of ¹²⁵ICSF-1 at various concentrations was saturated at about 3 ng/ml. The binding sites of ¹²⁵ICSF-1 were sensitive to trypsin but not to DNase or RNase treatment. At 37 °C, the trypsin-treated cells regenerated more than 90% of their original binding sites within 12 h. Whereas more than 97% of these alveolar macrophages were phagocytic and esterase-positive, autoradiographic studies showed that only 10–31 % of them were capable of binding to ¹²⁵ICSF-1. These results indicate that the frequencies of CSF-1-binding cells and alveolar macrophage colony-forming cells (AL-CFC) are closely correlated, but no causal relationship has been established.     

Binding, internalization and degradation of colony-stimulating factor by peritoneal exudate macrophages

Iodinated colony-stimulating factor produced by L-cells (¹²⁵I-CSF-1) binds specifically to murine peritoneal exudate macrophages. At 37°C, the cell-bound ¹²⁵I-CSF-1 was internalized and degraded very rapidly, with the appearance of radioactive iodotyrosine in the medium. At 0°C, the cell-bound ¹²⁵I-CSF-1 was not internalized and degraded, nor did it dissociate from the membrane. The internalization and degradation at 37°C could be blocked or reduced by the presence of phenylglyoxal, methylamine and NH₄Cl. The chemical nature of the CSF-1 binding site is polypeptide as judged by its sensitivity to trypsin treatment. After the binding and degradation of unlabeled CSF-1, the exudate cells were no longer able to rebind freshly added ¹²⁵I-CSF-1, indicating the removal of CSF-1 binding site. The binding capacity of these cells, however, could be restored by prolonged incubation at 37°C but not at 0°C in culture medium containing fetal calf serum.     

In situ heart rate and activity of incubating Adélie penguins (Pygoscelis adeliae)

Summary Heart rates and activity were monitored over 24 h in unrestrained, incubating Adélie penguins (Pygoscelis adeliae) exposed to natural conditions in the colony. Heart rate (HR in bpm) increased linearly with wind speed (w; range 0–19 m/s): HR = 85.8+1.35 w, but was unrelated (P>0.05) to temperature (-2.5°–6°C), humidity (37%–100%) cloud cover (0–8/8) and estimated solar radiation (0–12). Wind-induced heat loss was apparently compensated to a large degree by increased metabolic activity. Activity (A) measured as frequency of standing per hour, decreased linearly with temperature (t) and wind speed (w): A = 1.651–0.033w–0.090t. After correcting for meteorological influences, heart rate and bird activity showed no diurnal periodicity. When incubating, metabolism and activity of Adélie penguins appear to be mainly governed by climatic variations.     

Effects of acute temperature or salinity stress on the immune response in sea cucumber, Apostichopus japonicus

Invertebrates are increasingly raised in mariculture, where it is important to monitor immune function and to minimize stresses that could suppress immunity. The activities of phagocytosis, superoxide dismutase (SOD), catalase (CAT), myeloperoxidase (MPO), and lysozyme (LSZ) were measured to evaluate the immune capacities of the sea cucumber, Apostichopus japonicus, to acute temperature changes (from 12 °C to 0 °C, 8 °C, 16 °C, 24 °C, and 32 °C for 72 h) and salinity changes (from 30‰ to 20‰, 25‰, and 35‰ for 72 h) in the laboratory. Phagocytosis was significantly affected by temperature increases in 3 h, and by salinity (25‰ and 35‰) changes in 1 h. SOD activities decreased significantly in 0.5 h to 6 h samples at 24 °C. At 32 °C, SOD activities decreased significantly in 0.5 h and 1 h exposures, and obviously increased for 12 h exposure. CAT activities decreased significantly at 24 °C for 0.5 h exposure, and increased significantly at 32 °C in 3 h to 12 h exposures. Activities of MPO increased significantly at 0 °C in 0.5 h to 6 h exposures and at 8 °C for 1 h. By contrast, activities of MPO decreased significantly in 24 °C and 32 °C treatments. In elevated-temperature treatments, activities of LSZ increased significantly except at 32 °C for 6 h to 12 h exposures. SOD activity was significantly affected by salinity change. CAT activity decreased significantly after only 1 h exposure to salinity of 20‰. Activities of MPO and LSZ showed that A. japonicus tolerates limited salinity stress. High-temperature stress had a much greater effect on the immune capacities of A. japonicus than did low-temperature and salinity stresses.     

Purification and Properties of Cold-active Metalloprotease from Curtobacterium luteum and Effect of Culture Conditions on Production

Curtobacterium luteum, a gram-positive psychrotrophic bacterium, secreting an extracellular protease was isolated from the soil of Gangotri glacier, Western Himalaya. The maximum enzyme production was achieved when isolate was grown in a pH-neutral medium containing skim milk at 15oC over 120 hour. The metal ions such as Zn2+ and Cr2+ enhanced enzyme production. The specific activity of purified enzyme was 8090 u/mg after 34.1 fold purification. The 115 kD enzyme was a metalloprotease (activity inhibited by EDTA and EGTA) and showed maximum activity at 20oC and pH 7. The enzyme was active over a broad pH range and retained 84% of its original activity between pH 6–8. There was no loss in enzyme activity when exposed for 3 hours at 4oC-20oC. However, lost 65% of activity at 30oC, and was almost inactivated at 50oC, but was resistant to repeated freezing and thawing. The enzyme activity was stimulated by manganese ions; however, it was inactivated by copper ions.     

Formamide denaturation of chromosomal DNA for in situ hybridization and C-band preparation in the guinea pig, Cavia porcellus

Cytological preparations were incubated in 0.07 N NaOH at room temperature or 90% formamide (final salt concentration 2 × SSC) at either 65 °C or 37 °C for 2.5 h to denature guinea pig chromosomes. Chromosomes treated with NaOH or formamide at 65 °C showed a large amount of DNA loss, while chromosomes treated with formamide at 37 °C showed little or no DNA loss. Repeated sequences were isolated from guinea pig DNA and [³H]cRNA was transcribed with Escherichia coli RNA polymerase for in situ hybridization. Localization of the [³H]cRNA occurred in the centromeric regions and C-band positive short arms of almost all of the chromosomes in the NaOH preparations. Chromosomes treated with formamide at 65 °C showed the same grain distribution with a decrease in the number of grains/cluster. Slides incubated in formamide at 37 °C showed localization in only a few chromosomes and the number of grains/cluster was greatly diminished. Thermal denaturation of isolated chromatin indicated that incubation of chromosomes in formamide at 37 °C did not fully denature the DNA. C-bands could be induced by treating slides in formamide at either 65 °C or 37 °C when followed by a “reassociation” in 2 × SSC at 65 °C for 16 h. If the “reassociation” step was omitted, C-bands were found in the 65 °C formamide slides but not the 37 °C formamide slides.     

Binding of plasma fibronectin to the surfaces of BHK cells in suspension at 4 °C

Plasma fibronectin is shown by several different criteria to bind to suspended BHK cells if the binding incubations are carried out at 4 °C. In indirect immunofluorescence experiments, fibronectin bound to suspended BHK cells at 4 °C in a punctate distribution over the entire cell surfaces. Little binding, however, was detected on cells incubated with fibronectin at 37 °C. The fibronectin bound to the cells at 4 °C was functionally active, since these cells subsequently were able to spread on tissue culture dishes in protein-free medium, unlike cells preincubated with fibronectin at 37 °C or in the absence of fibronectin. Also, the cell surface receptors for soluble fibronectin and fibronectin-coated beads appeared to be similar, since cells preincubated with fibronectin at 4 °C subsequently bound fewer fibronectin-coated beads than control cells. In biochemical studies with radiolabeled fibronectin, binding of fibronectin to the cells was shown to increase with incubation time up to 4 h. In competition experiments with unlabeled fibronectin, 30% of the binding of radiolabeled fibronectin could be inhibited.