Multipotent human stromal cells improve cardiac function after myocardial infarction in mice without long-term engraftment

Signaling via the type 1 insulin-like growth factor receptor (IGF1R) confers resistance to EGF receptor (EGFR) inhibitors. It is plausible that reciprocal EGFR compensation could mediate resistance to IGF1R inhibition, prompting us to investigate effects of IGF1R depletion on EGFR signaling in breast cancer cells expressing relatively high (MDA-MB-468) or low (MCF7) EGFR. Transient IGF1R knockdown induced enhanced phosphorylation of the EGFR and its effectors JNK, ERKs and STAT5, but this did not prevent apoptosis induction and inhibition of clonogenic survival following IGF1R knockdown. We used IGF1R shRNA to induce chronic IGF1R depletion, and achieved stable gene silencing in MCF-7 cells; here, EGFR overexpression led to EGFR hyperphosphorylation, again without abrogating survival inhibition after IGF1R knockdown. In both cell lines, dual receptor knockdown prevented EGFR hyperphosphorylation, but induced no greater inhibition of clonogenic survival than IGF1R knockdown alone. These results suggest that the EGFR cannot compensate for IGF1R depletion, and are encouraging for the strategy of IGF1R targeting.     

Survival of Pacific oyster, Crassostrea gigas, oocytes in relation to intracellular ice formation

The effect of IIF in Pacific oyster oocytes was studied using cryo and transmission electron microscopy (TEM). The viability of oocytes at each step of a published cryopreservation protocol was assessed in an initial experiment. Two major viability losses were identified; one when oocytes were cooled to −35 °C and the other when oocytes were plunged in liquid nitrogen. Although the cryomicroscope showed no evidence of IIF in oocytes cooled with this protocol, TEM revealed that these oocytes contained ice crystals and were at two developmental stages when frozen, prophase and metaphase I. To reduce IIF, the effect of seven cooling programmes involving cooling to −35 or −60 °C at 0.1 or 0.3 °C min⁻¹ and holding for 0 or 30 min at −35 or −60 °C was evaluated on post-thaw fertilization rate of oocytes. Regardless of the cooling rate or holding time, the fertilization rate of oocytes cooled to −60 °C was significantly lower than that of oocytes cooled to −35 °C. The overall results indicated that observations of IIF obtained from cryomicroscopy are limited to detection of larger amounts of ice within the cells. Although the amount of cellular ice may have been reduced by one of the programmes, fertilization was reduced significantly; suggesting that there is no correlation between the presence of intracellular ice and post-thaw fertilization rate. Therefore, oyster oocytes may be more susceptible to the effect of high solute concentrations and cell shrinkage than intracellular ice under the studied conditions.     

Calorimetric measurement of water transport and intracellular ice formation during freezing in cell suspensions

The current study presents a new and novel analysis of heat release signatures measured by a differential scanning calorimeter (DSC) associated with water transport (WT), intracellular ice formation (IIF) and extracellular ice formation (EIF). Correlative cryomicroscopy experiments were also performed to validate the DSC data. The DSC and cryomicroscopy experiments were performed on human dermal fibroblast cells (HDFs) at various cytocrit values (0–0.8) at various cooling rates (0.5–250 °C/min). A comparison of the cryomicroscopy experiments with the DSC analysis show reasonable agreement in the water transport (cellular dehydration) and IIF characteristics between both the techniques with the caveat that IIF measured by DSC lagged that measured by cryomicroscopy. This was ascribed to differences in the techniques (i.e. cell vs. bulk measurement) and the possibility that not all IIF is associated with visual darkening. High and low rates of 0.5 °C/min and 250 °C/min were chosen as HDFs did not exhibit significant IIF or WT at each of these extremes respectively. Analysis of post-thaw viability data suggested that 10 °C/min was the presumptive optimal cooling rate for HDFs and was independent of the cytocrit value. The ratio of measured heat values associated with IIF (q_IIF) to the total heat released from both IIF and water transport or from the total cell water content in the sample (q_CW) was also found to increase as the cooling rate was increased from 10 to 250 °C/min and was independent of the sample cytocrit value. Taken together, these observations suggest that the proposed analysis is capable of deconvolving water transport and IIF data from the measured DSC latent heat thermograms in cell suspensions during freezing.     

Experimental study of intracellular ice growth in human umbilical vein endothelial cells

Study of the intracellular ice formation (IIF) and growth is essential to the mechanistic understanding of cellular damage through freezing. In the aid of high speed and high resolution cryo-imaging technology, the transient intracellular ice formation and growth processes of the attached human umbilical vein endothelial cells (HUVEC) were successfully captured during freezing. It was found that the intracellular ice nucleation site was on the cell membrane closer to the nucleus. The ice growth was directional and toward the nucleus, which covered the whole nucleus before growing into the cytoplasm. The crystal growth rate in the nucleus was much larger than that in the cytoplasm, and its morphology was influenced by the cooling rate. During the thawing process, small crystals fused into larger ones inside the nucleus. Moreover, the cumulative fraction of the HUVEC with IIF was mainly dependent on the cooling rate not the confluence of the cells attached.     

A microscale model for prediction of breast cancer cell damage during cryosurgery

The morphology of cancerous breast tissue is characterized by tightly packed groups of small malignant cells, as found in most duct cell carcinoma. This special structure affects the osmotic responses of the cells to freezing and hence their probability of damage from cellular dehydration or intracellular ice formation. A mathematical model has been developed to study the microscale damage to these breast cancer cells during cryosurgery by accounting for their special structure. The model is based on a spherical unit comprised of an extracellular region that surrounds several layers of cancer cells, as experimentally observed of breast duct cell carcinoma by other researchers. Temperature transients in the breast cancer undergoing cryosurgery are calculated numerically using the Pennes equation. When subjected to various thermal histories, both cellular dehydration and intracellular ice formation in the unit structure are examined by considering the cell-to-cell contact and water transport at the microscale level. It is found that the cells in the inner layers hardly dehydrated while those in the outermost layer do greatly. The results help interpret the previously observed experimental phenomena that breast cancer tissues exhibit intracellular ice formation even at a slow cooling rate of -3 degrees C/min. In the attempt to better define an optimal procedure for breast cancer cryosurgery, various freezing protocols are simulated. The constant heat flux protocol induces greater cellular dehydration and higher intracellular ice formation probability simultaneously compared to the other protocols studied.     

Investigating cryoinjury using simulations and experiments. 1: TF-1 cells during two-step freezing (rapid cooling interrupted with a hold time)

There is significant interest in designing a cryopreservation protocol for hematopoietic stem cells (HSC) which does not rely on dimethyl sulfoxide (Me₂SO) as a cryoprotectant. Computer simulations that describe cellular osmotic responses during cooling and warming can be used to optimize the viability of cryopreserved HSC; however, a better understanding of cellular osmotic parameters is required for these simulations. As a model for HSC, the erythroleukemic human cell line TF-1 was used in this study. Simulations, based on the osmotic properties of TF-1 cells and on the solution properties of the intra- and extracellular compartments, were used to interpret cryoinjury associated with a two-step cryopreservation protocol. Calculated intracellular supercooling was used as an indicator of cryoinjury related to intracellular ice formation. Simulations were applied to the two-step cooling protocol (rapid cooling interrupted with a hold time) for TF-1 cells in the absence of Me₂SO or other cryoprotectants and optimized by minimizing the indicator of cryoinjury. A comparison of simulations and experimental measurements of membrane integrity supports the concept that, for two-step cooling, increasing intracellular supercooling is the primary contributor to potential freezing injury due to the increase in the likelihood of intracellular ice formation. By calculating intracellular supercooling for each step separately and comparing these calculations with cell recovery data, it was demonstrated that it is not optimal simply to limit overall supercooling during two-step freezing procedures. More aptly, appropriate limitations of supercooling differ from the first step to the second step. This study also demonstrates why high cell recovery after cryopreservation could be achieved in the absence of traditional cryoprotectants.     

Differential Modulation of 7-Ketocholesterol Toxicity Against PC12 Cells by Calmodulin Antagonists and Ca<Superscript>2+</Superscript> Channel Blockers

The present study assessed the influence of intracellular Ca²⁺ and calmodulin against the neurotoxicity of oxysterol 7-ketocholesterol in relation to the mitochondria-mediated cell death process and oxidative stress in PC12 cells. Calmodulin antagonists calmidazolium and W-7 prevented the 7-ketocholesterol-induced mitochondrial damage, leading to caspase-3 activation and cell death, whereas Ca²⁺ channel blocker nicardipine, mitochondrial Ca²⁺ uptake inhibitor ruthenium red, and cell permeable Ca²⁺ chelator BAPTA-AM did not reduce it. Exposure of PC12 cells to 7-ketocholesterol caused elevation of intracellular Ca²⁺ levels. Unlike cell injury, calmodulin antagonists, nicardipine, and BAPTA-AM prevented the 7-ketocholesterol-induced elevations of intracellular Ca²⁺ levels. The results show that the cytotoxicity of 7-ketocholesterol seems to be modulated by calmodulin rather than changes in intracellular Ca²⁺ levels. Calmodulin antagonists may prevent the cytotoxicity of 7-ketocholesterol by suppressing the mitochondrial permeability transition formation, which is associated with the increased formation of reactive oxygen species and the depletion of GSH.     

Change of supercooling capability in solutions containing different kinds of ice nucleators by flavonol glycosides from deep supercooling xylem parenchyma cells in trees

Deep supercooling xylem parenchyma cells (XPCs) in Katsura tree contain flavonol glycosides with high supercooling-facilitating capability in solutions containing the ice nucleation bacterium (INB) Erwinia ananas, which is thought to have an important role in deep supercooling of XPCs. The present study, in order to further clarify the roles of these flavonol glycosides in deep supercooling of XPCs, the effects of these supercooling-facilitating (anti-ice nucleating) flavonol glycosides, kaempferol 3-O-β-d-glucopyranoside (K3Glc), kaempferol 7-O-β-d-glucopyranoside (K7Glc) and quercetin 3-O-β-d-glucopyranoside (Q3Glc), in buffered Milli-Q water (BMQW) containing different kinds of ice nucleators, including INB Xanthomonas campestris, silver iodide and phloroglucinol, were examined by a droplet freezing assay. The results showed that all of the flavonol glycosides promoted supercooling in all solutions containing different kinds of ice nucleators, although the magnitudes of supercooling capability of each flavonol glycoside changed in solutions containing different kinds of ice nucleators. On the other hand, these flavonol glycosides exhibited complicated nucleating reactions in BMQW, which did not contain identified ice nucleators but contained only unidentified airborne impurities. Q3Glc exhibited both supercooling-facilitating and ice nucleating capabilities depending on the concentrations in such water. Both K3Glc and K7Glc exhibited only ice nucleation capability in such water. It was also shown by an emulsion freezing assay in BMQW that K3Glc and Q3Glc had no effect on homogeneous ice nucleation temperature, whereas K7Glc increased ice nucleation temperature. The results indicated that each flavonol glycoside affected ice nucleation by very complicated and varied reactions. More studies are necessary to determine the exact roles of these flavonol glycosides in deep supercooling of XPCs in which unidentified heterogeneous ice nucleators may exist.     

Rabbit distal colon epithelium: I. Isolation and characterization of basolateral plasma membrane vesicles from surface and crypt cells

Summary A method has been developed for the simultaneous isolation of basolateral plasma membrane vesicles from surface and crypt cells of rabbit distal colon epithelium by sequential use of differential sedimentation, isopycnic centrifugation and Ficoll 400 barrier centrifugation. The protein yield was high (total 0.81 mg/g mucosa) and surface and crypt cell-derived basolateral membrane fractions have been purified 34- and 9-fold with respect to the homogenate. The pattern of marker enzyme enrichments revealed only minor contamination by subcellular organelles. Latency of ouabain-sensitive (Na⁺, K⁺)-ATPase activity prior and after trypsin treatment of membranes indicated a vesicle configuration of sealed right side-out: sealed inside-out: leaky of approximately 211. The presence of sealed vesicles was also evident from the osmotic sensitivity of thed-[1-¹⁴C] mannitol equilibrium space determined with either fraction. Although considerably different in protein profile, surface and crypt basolateral membranes were similar in cholesterol to phospholipid molar ratio and membrane fluidity as determined by steady-state fluorescence polarization.Stopped-flow light scattering experiments revealed a rather low water permeability of the membranes with a permeability coefficient of 6 m/sec at 35°C, which is one order of magnitude lower than reported for small intestinal plasma membranes. Both membrane fractions have been shown to effectively generate outward uphill potassium ion gradients, a process that is energized by ATP and inhibited by the membrane-permeant cardiacglycoside digitoxin. These characteristics are consistent with the activity of a (Na⁺, K⁺) pump operating in inside-out vesicles.     

Differential Involvement of Intracellular Ca2+ in 1-Methyl-4-phenylpyridinium- or 6-Hydroxydopamine-Induced Cell Viability Loss in PC12 Cells

1-Methyl-4-phenylpyridinium (MPP⁺) or 6-hydroxydopamine (6-OHDA) caused a nuclear damage, the mitochondrial membrane permeability changes, leading to the cytochrome c release and caspase-3 activation, the formation of reactive oxygen species and the depletion of GSH in PC12 cells. Nicardipine (a calcium channel blocker), EGTA (an extracellular calcium chelator), BAPTA-AM (a cell permeable calcium chelator) and calmodulin antagonists (W-7 and calmidazolium) attenuated the MPP⁺-induced mitochondrial damage and cell death. In contrast, the compounds did not reduce the toxicity of 6-OHDA. Treatment with MPP⁺ or 6-OHDA evoked the elevation of intracellular Ca²⁺ levels. Unlike cell injury, addition of nicardipine, BAPTA-AM and calmodulin antagonists prevented the elevation of intracellular Ca²⁺ levels due to both toxins. The results show that the MPP⁺-induced formation of the mitochondrial permeability transition seems to be mediated by elevation of intracellular Ca²⁺ levels and calmodulin action. In contrast, the 6-OHDA-induced cell death seems to be mediated by Ca²⁺-independent manner.     

Antioxidant Effect of Phenelzine on MPP+-Induced Cell Viability Loss in Differentiated PC12 Cells

Phenelzine, deprenyl, and antioxidants (SOD, catalase, ascorbate, or rutin) reduced the loss of cell viability in differentiated PC12 cells treated with 250 M MPP⁺, whereas N-acetylcysteine and dithiothreitol did not inhibit cell death. Phenelzine reduced the condensation and fragmentation of nuclei caused by MPP⁺ in PC12 cells. Phenelzine and deprenyl prevented the MPP⁺-induced decrease in mitochondrial membrane potential, cytochrome c release, formation of reactive oxygen species, and depletion of GSH in PC12 cells. Phenelzine revealed a scavenging action on hydrogen peroxide and reduced the hydrogen peroxide–induced cell death in PC12 cells, whereas deprenyl did not depress the cytotoxic effect of hydrogen peroxide. Both compounds reduced the iron and EDTA-mediated degradation of 2-deoxy-d-ribose degradation. The results suggest that phenelzine attenuates the MPP⁺-induced viability loss in PC12 cells by reducing the alteration of mitochondrial membrane permeability that seems to be mediated by oxidative stress.