In vivo gene transfer between interacting human osteosarcoma cell lines is associated with acquisition of enhanced metastatic potential

We report here in vivo gene transfer between cancer cells is associated with acquisition of high metastatic behavior. The 143B‐GFP cell line with high metastatic potential and the MNNG/HOS‐RFP cell line with low metastatic potential, both derived from the TE85 human osteosarcoma cell line, were either co‐transplanted or transplanted alone in the tibia in nude mice. Upon mixed transplantation of the two differently labeled sublines, resulting metastatic colonies are single colored either red or green, thereby demonstrating their clonality and enabling facile color‐coded quantification. When MNNG/HOS‐RFP and 143B‐GFP were co‐transplanted in the tibia, the number of lung metastases of MNNG/HOS‐RFP increased eight‐fold compared to MNNG/HOS‐RFP transplanted alone (P < 0.01). In contrast, no enhancement of MNNG/HOS‐RFP metastases occurred when MNNG/HOS‐RFP and 143B‐GFP were transplanted separately in the right and left tibiae, respectively. This result suggests that the presence of 143B‐GFP increased the metastatic potential of MNNG/HOS‐RFP within the mixed tumor. We observed transfer of the Ki‐ras gene from 143B‐GFP to MNNG/HOS‐RFP after they were co‐implanted suggesting the Ki‐ras played a role in increasing the metastatic potential of MNNG/HOS‐RFP in the presence of 143B‐GFP. These data suggest the possible role of in vivo gene transfer in enhancing the metastatic potential of cancer cells. The data also further demonstrated the power of color‐coded imaging to visualize cancer‐cell/cancer‐cell interactions in vivo. J. Cell. Biochem. 108: 362–367, 2009. © 2009 Wiley‐Liss, Inc.     

Energy restriction with protein restriction increases basal metabolism and meal-induced thermogenesis in rats

We previously observed an increased sympathetic nervous system (SNS) activity that was partly responsible for a defect in the insulin secretion response to glucose after postweaning protein-energy restriction (PER) in female rats. These results, together with other data on low-protein feeding, suggested that a low protein-to-energy ratio (P/E) in the diet could stimulate energy expenditure (EE), but direct measurements of EE have never been reported under conditions of PER. The goal of the present study was thus to quantify the changes induced by PER to body composition, the various parameters of EE, and plasma triiodothyronine levels. PER induced severe growth retardation, but the subcutaneous white and interscapular brown adipose tissue masses were preserved. Basal metabolism, meal-induced thermogenesis, and triiodothyronine levels were increased, but substrate utilization by the working muscles was unaffected. Meal-induced thermogenesis was increased by spontaneous activity in PER rats only. These results suggest that rats adapt to a low P/E in the diet by burning part of their excess nonprotein energy and storing the remaining excess in subcutaneous adipose tissue.     

Effects of diethyldithiocarbamate and endogenous polyamine content on cellular responses to hydrogen peroxide cytotoxicity.

In exponential-phase Chinese-hamster cells, 0.1 mM-diethyldithiocarbamate (DDC) afforded greater than 1 log survival protection to cultures treated before and during exposure to 1 mM-H2O2. Both DDC and H2O2 treatment stimulated the activity of ornithine decarboxylase (ODC), the first enzyme in polyamine synthesis, within 4 h of exposure. DDC, and to a lesser degree H2O2, also stimulated the activity of spermidine N1-acetyltransferase (SAT), the rate-limiting enzyme in polyamine catabolism. The increase in SAT activity, after exposure to DDC or another stress (heat shock), was inhibited in cells depleted of putrescine and spermidine by alpha-difluoromethylornithine (DFMO), the enzyme-activated suicide inhibitor of ODC. Pretreatment with DFMO or heat shock also induced resistance to H2O2 cytotoxicity. Since SAT activity is low in resting cells, yet stimulation of enzyme activity depends on endogenous spermidine pools, these results suggest that the expression of SAT activity occurs by a mechanism involving a stress-dependent displacement of spermidine into a new intracellular compartment. The stimulation of ODC and SAT activities does not appear to be a necessary component of the mechanism by which DDC protects cells from H2O2 cytotoxicity, although spermidine displacement may be a common facet of the cellular response to stress.     

Pulmonary compliance values provide prognosis in mechanically ventilated patients--a randomized prospective study

Our aim was to evaluate the influence of static pulmonary compliance (Cst) on the choice of Mechanical Ventilation(MV) method and treatment outcome. A prospective, randomized trial conducted out at the multidisciplinary Intensive Care Unit (ICU) included 387 patients, randomized in two groups: noninvasive MV group and invasive MV group. Furthermore, each group was divided in two groups: Cst < or = 0.025 and Cst > 0.025 L/cm H2O. In patients with Cst > 0.025 L/cm H2O MV duration, noninvasive vs invasive, was 92 vs 114 h, p = 0.039, time spent in ICU 118 vs 164 h, p = 0.004. In patients with Cst < or = 0.025, MV duration was 141 vs 189 h, p < 0.001, time spent in ICU 190 vs 246 h, p = 0.001, all patients were intubated. Need for tracheostomy was 6 (11%) vs 39 (46%) patients, p = 0.005, and ICU mortality was 15 (26%) vs 21 (25%) patients. Statistical significance in favor of noninvasive method was confirmed in patients with Cst > 0.025 L/cm H2O in MV duration, time spent in ICU, need for tracheostomy and intubation rate. In the group with Cst < or = 0.025 no significant difference in treatment failure was recorded between the two MV methods.     

Meal cysteine improves postprandial glucose control in rats fed a high-sucrose meal

Whey protein, particularly the alpha-lactalbumin fraction, are rich in cysteine (cys) and could therefore favor postprandial glucose homeostasis by a glutathione-mediated effect. This work investigates the effects of the ingestion of an alpha-lactalbumin-rich whey concentrate (alpha-LAC) during a high-sucrose (HS) meal on postprandial glucose homeostasis in healthy rats. In the first experiment, rats received an HS meal containing 14% protein, in which the protein source was either alpha-LAC (HS(a)) or total milk proteins, alone (HS(0)) or supplemented with 17 mg (HS(1)) or 59 mg (HS(2)) of N-acetylcysteine (NAC). This resulted in a total cys content 3.6-fold higher in the HS(1) and HS(a) meals and 12-fold higher in the HS(2) meal, when compared to the HS(0) meal. Postprandial parameters were monitored for 3 h after ingestion of the meal. The same measurements were performed on rats injected with 4 mmol/kg of buthionine sulfoximine (BSO), a specific inhibitor of glutathione synthesis. Increasing the meal's cys content dose-dependently reduced both postprandial glucose and insulin (P<.05). The inhibition of glutathione synthesis with BSO injection abrogated the beneficial effects of NAC supplementation on postprandial glucose response but did not affect those of alpha-LAC. These results show that (1) the substitution of alpha-LAC for total milk protein reduces glucose response, as does the addition of a cys donor to the meal, (2) but contrary to those of a simple cys donor, the beneficial effects of alpha-LAC are not entirely mediated by glutathione synthesis, suggesting additional mechanisms.     

Water-deficit tolerance induction during germination of Jalo Precoce bean (Phaseolus vulgaris L.) cultivar

Bean (Phaseolus vulgaris L.) cultivars are susceptible to water stress, especially during germination. Salicylic acid and thermal shock improve tolerance level in this plant to different kinds of stresses. However, in the present work, effects of cold shock and salicylic acid alone or combined on germinating beans under imposed water deficit were evaluated. Bean seeds from the Jalo Precoce cultivar were imbibed either in water or in salicylic acid for 24 h. After treatment, lots were divided and exposed to cold shock (7 °C) for another 24 h. The seeds were then placed on paper rolls imbibed with different concentrations of mannitol to impose a water deficit on them. They were evaluated for germination; seedling vigour; dry and total weights and length of shoot root; superoxide dismutase activity and proline content. Proline accumulates as a response to water-deficit stress, and SA alone or combined with cold shock improves this response. The use of SA increases many of the physiological variables than cold shock and decreases the antioxidant activity of SOD.     

Viruses, plasmids and other genetic elements of thermophilic and hyperthermophilic Archaea

We review and update the work on genetic elements, e.g., viruses and plasmids (excluding IS elements and transposons) in the kingdom Crenarchaeota (Thermoproteales and Sulfolobales) and the orders Thermococcales and Thermoplasmales in the kingdom Euryachaeota of the archael domain, including unpublished data from our laboratory. The viruses of Crenarchaeota represent four novel virus families. The Fuselloviridae represented by SSV1 of S. shibatae and relatives in other Sulfolobus strains have the form of a failed spindle. The envelope is highly hydrophobic. The DNA is double-stranded and circular. Members of this group have also been found in Methanococcus and Haloarcula. The Lipothrixviridae (e.g., T TV1 to 3) have the form of flexible filaments. They have a core containing linear double-stranded DNA and DNA-binding proteins which is wrapped into a lipid membrane. The ‘Bacilloviridae’ (e.g., TTV4 and SIRV) are stiff rods lacking this membrane, but also featuring linear double-stranded DNA and DNA-binding proteins. Both virus type carry on both ends structures involved in the attachment to receptors. Both types are represented in Thermoproteus and Sulfolobus. The droplet-formed novel Sulfolobus virus SNDV represents the ‘Guttaviridae’ containing circular double-stranded DNA. Though head and tail viruses distantly resembling T phages or lambdoid phages were seen electronmicroscopically in solfataric water samples, no such virus has so far been isolated. SSV1 is temperate, TTV1 causes lysis after induction, the other viruses found so far exist in carrier states. The hosts of all but TTV1 survive virus production. We discuss the implications of the nature of these viruses for understanding virus evolution. The plasmids found so far range in size from 4.5 kb to about 40 kb. Most of them occur in high copy number, probably due to the way of their detection. Most are cryptic, pNOB8 is conjugative, the widespread pDL10 alleviates in an unknown way autotrophic growth of its host Desulfurolobus by sulfur reduction. The plasmid pTIK4 appears to encode a killer function. pNOB8 has been used as a vector for the transfer of the lac S (β-galactosidase) gene into a mutant of S. solfataricus.     

Manganese porphyrin, MnTE-2-PyP5+, Acts as a pro-oxidant to potentiate glucocorticoid-induced apoptosis in lymphoma cells

Using current chemotherapy protocols, over 55% of lymphoma patients fail treatment. Novel agents are needed to improve lymphoma survival. The manganese porphyrin, MnTE-2-PyP(5+), augments glucocorticoid-induced apoptosis in WEHI7.2 murine thymic lymphoma cells, suggesting that it may have potential as a lymphoma therapeutic. However, the mechanism by which MnTE-2-PyP(5+) potentiates glucocorticoid-induced apoptosis is unknown. Previously, we showed that glucocorticoid treatment increases the steady state levels of hydrogen peroxide ([H(2)O(2)](ss)) and oxidizes the redox environment in WEHI7.2 cells. In the current study, we found that when MnTE-2-PyP(5+) is combined with glucocorticoids, it augments dexamethasone-induced oxidative stress however, it does not augment the [H(2)O(2)](ss) levels. The combined treatment depletes GSH, oxidizes the 2GSH:GSSG ratio, and causes protein glutathionylation to a greater extent than glucocorticoid treatment alone. Removal of the glucocorticoid-generated H(2)O(2) or depletion of glutathione by BSO prevents MnTE-2-PyP(5+) from augmenting glucocorticoid-induced apoptosis. In combination with glucocorticoids, MnTE-2-PyP(5+) glutathionylates p65 NF-κB and inhibits NF-κB activity. Inhibition of NF-κB with SN50, an NF- κB inhibitor, enhances glucocorticoid-induced apoptosis to the same extent as MnTE-2-PyP(5+). Taken together, these findings indicate that: 1) H(2)O(2) is important for MnTE-2-PyP(5+) activity; 2) Mn-TE-2-PyP(5+) cycles with GSH; and 3) MnTE-2-PyP(5+) potentiates glucocorticoid-induced apoptosis by glutathionylating and inhibiting critical survival proteins, including NF-κB. In the clinic, over-expression of NF-κB is associated with a poor prognosis in lymphoma. MnTE-2-PyP(5+) may therefore, synergize with glucocorticoids to inhibit NF-κB and improve current treatment.