Apoptosis in Postmortal Tissues of Goat Spinal Cords and Survival of Resident Neural Progenitors.

Growing demand for therapeutic tissue repair recurrently focusses scientists' attention on critical assessment of postmortal collection of live cells, especially stem cells. Our study aimed to assess the survival of neuronal progenitors in postmortal spinal cord and their differentiation potential. Postmortal samples of spinal cords were obtained from human-sized animals (goats) at 6, 12, 24, 36, and 54 h after slaughter. Samples were studied by immunohistology, differentiation assay, Western blot and flow cytometry for the presence and location of GD2-positive neural progenitors and their susceptibility to cell death. TUNEL staining of the goat spinal cord samples over 6-54 h postmortem revealed no difference in the number of positive cells per cross-section. Many TUNEL-positive cells were located in the gray commissure around the central canal of the spinal cord; no increase in TUNEL-positive cells was recorded in either posterior or anterior horns of the gray matter where many GD2-positive neural progenitors can be found. The active caspase 3 amount as measured by Western blot at the same intervals was moderately increasing over time. Neuronal cells were enriched by magnetic separation with antibodies against CD24; among them, the GD2-positive neural progenitor subpopulation did not overlap with apoptotic cells having high pan-caspase activity. Apoptotic cell death events are relatively rare in postmortal spinal cords and are not increased in areas of the neural progenitor cell's location, within measured postmortal intervals, or among the CD24/GD2-positive cells. Data from our study suggest postmortal spinal cords as a valuable source for harvesting highly viable allogenic neural progenitor cells.

Thermospheric parameters contribution to the formation of Yakutsk F2-layer diurnal summer time anomaly.

The role of thermospheric neutral composition in the formation of the Yakutsk diurnal summer time foF2 anomaly is analyzed. Ionospheric stations inside and outside the anomaly area are considered. The effect of neutral composition in foF2 is the most noticeable around noontime hours. The difference between observed noontime foF2 in two areas is significant at the 99.9% confidence level both for monthly median and individual days. The inferred from ionosonde observations and Swarm neutral gas density thermospheric parameters indicate a significant difference between two areas. The inferred exospheric temperature, Tex at Magadan (inside the anomaly area) is significantly larger than Tex at Tunguska (outside the anomaly area). On the contrary, the inferred atomic oxygen [O] at Tunguska is significantly larger than at Magadan. Different [O] abundance in the two areas is the main reason of the observed difference in noontime foF2 values. Vertical plasma drift depending on magnetic declination, D is the only process responsible for the difference between nighttime foF2 at Tunguska and Magadan. A possible mechanism of the revealed difference in thermospheric parameters inside and outside the anomaly area is discussed.

Influence of the perfusion bioreactor on Stratified and Distributed approaches for multilayered tissue engineering on biodegradable scaffolds.

Despite wide use and approval of poly lactic-coglycolic acid (PLGA) for surgical applications, there have been very few studies on tissue constructions that mimic physiological multilayer structures by combining PLGA scaffolds with tissue engineering. In our study, we developed a bioreactor system to maintain, and to train two types of three-layered vascular-like structures. Then we examined how the perfusion conditions and different tissue engineering approaches affected the formation of the layered structure and degradation of the PLGA scaffolds. In the proposed Distributed Method, the cells were seeded layer by layer on a single scaffold, using spheroids bigger than scaffold fiber gaps and we achieved the higher cell density compared with the Stratified Method where we stacked three PLGA sheets seeded with individual vascular cell types. At the histological level, scaffold degradation was more prominent in the bioreactor compared to the same time interval in vivo. In addition, the faster flow accelerated the decomposition of PLGA fibers. Moreover, bioreactor perfusion culture at lower flow rates could balance cell adhesion and survival, improve the cell density and promote self-organization of multilayer structure with desirable rate of PLGA scaffolds degradation.

Dynamic flow priming programs allow tuning up the cell layers properties for engineered vascular graft.

Tissue engineered vascular grafts (TEVG) are potentially clear from ethical and epidemiological concerns sources for reconstructive surgery for small diameter blood vessels replacement. Here, we proposed a novel method to create three-layered TEVG on biocompatible glass fiber scaffolds starting from flat sheet state into tubular shape and to train the resulting tissue by our developed bioreactor system. Constructed tubular tissues were matured and trained under 3 types of individual flow programs, and their mechanical and biological properties were analyzed. Training in the bioreactor significantly increased the tissue burst pressure resistance (up to 18 kPa) comparing to untrained tissue. Fluorescent imaging and histological examination of trained vascular tissue revealed that each cell layer has its own individual response to training flow rates. Histological analysis suggested reverse relationship between tissue thickness and shear stress, and the thickness variation profiles were individual between all three types of cell layers. Concluding: a three-layered tissue structure similar to physiological can be assembled by seeding different cell types in succession; the following training of the formed tissue with increasing flow in a bioreactor is effective for promoting cell survival, improving pressure resistance, and cell layer formation of desired properties.

Lung cancer increases H2O2 concentration in the exhaled breath condensate, extent of mtDNA damage, and mtDNA copy number in buccal mucosa.

We have shown that the H2O2 concentration in exhaled breath condensate (EBC) in lung cancer patients increases significantly compared to the EBC of healthy people and revealed the correlation between the H2O2 level in the EBC and amount of mtDNA damage in buccal mucosa cells. The H2O2 hyper-production may trigger mitochondrial biogenesis, thereby resulting in an increase in mtDNA copy number. However, we did not observe a significant difference in the studied parameters between smokers and non-smokers. Overall, our data suggest that H2O2 concentration in the EBC, the extent of mtDNA damage, and mtDNA copy number in buccal mucosa could be potential as an early diagnostic marker of lung cancer.

Daytime mid-latitude F2-layer Q-disturbances: A formation mechanism.

Negative and positive near noontime prolonged (≥3 hours) F2-layer Q-disturbances with deviations in NmF2 > 35% occurred at Rome have been analyzed using aeronomic parameters inferred from fp180 (plasma frequency at 180 km height) and foF2 observations. Both types of NmF2 perturbations occur under quiet (daily Ap < 15 nT) geomagnetic conditions. Day-to-day atomic oxygen [O] variations at F2-region heights specify the type (positive or negative) of Q-disturbance. The [O] concentration is larger on positive and is less on negative Q-disturbance days compared to reference days. This difference takes place not only on average but for all individual Q-disturbances in question. An additional contribution to Q-disturbances formation is provided by solar EUV day-to-day variations. Negative Q-disturbance days are characterized by lower hmF2 while positive - by larger hmF2 compared to reference days. This is due to larger average Tex and vertical plasma drift W on positive Q-disturbance days, the inverse situation takes place for negative Q-disturbance days. Day-to-day changes in global thermospheric circulation may be considered as a plausible mechanism. The analyzed type of F2-layer Q-disturbances can be explained in the framework of contemporary understanding of the thermosphere-ionosphere interaction based on solar and geomagnetic activity as the main drivers.

Longitudinal variations in thermospheric parameters under summer noontime conditions inferred from ionospheric observations: A comparison with empirical models.

Longitudinal variations in the thermospheric neutral composition ([O] and [N2]) and exospheric temperature Tex have been inferred from June monthly median noontime foF1 and foF2 observations at mid-latitudes to check for consistency with empirical MSIS models. In general, a similarity in longitudinal variations has been demonstrated, and this is interesting, as similar variations were obtained with very different methods and different data sources. Both inferred and MSISE-00 modelled height-integrated O/N2 ratios are comparable to TIMED/GUVI observations only under solar minimum conditions but differ substantially under high solar activity. The retrieved height-integrated O/N2 ratio longitudinal variations are small (∼15%) in comparison to the observed NmF2 variations under high solar activity. The height-integrated O/N2 ratio cannot be incorporated into the F2-layer formation mechanism; therefore, such observations cannot be used for any quantitative interpretation of NmF2 variations.

Cell penetrating caspase substrates promote survival of the transplanted cells.

OBJECTIVE: Cell survival in critical post-transplantation period is challenged by inflammation, lack of vascularization, and insufficient cell attachment anchoring. Temporally blocking cell death may increase cell survival, but it is important to possess no risks of sustained cell death signal blocking and possible malignant transformations. Regarding apoptotic cell death, multi-micromolar overloading the cell with competitive caspase substrates delays the effects of actual downstream enzyme activation processing. Later, when introduced substrate is consumed, and the caspase activation stimuli may still be present, the apoptotic cell death can proceed normally. RESULTS: Here we studied several synthetic peptides comprising from effector caspase activational cleavage sequences fused with various internalization motifs. Designed peptides showed rapid and efficient internalization into cultured neuroblast cells comparing to non-fused cleavage sequences as measured by cytofluorimetry and confirmed by mass spectrometry. Pretreatment with selected peptides protected the cells from several apoptogenic stimuli in vitro, as well as improved survival of syngeneic immortalized Schwann cells during transplantation in vivo.

Long-term safety of the carbon fiber as an implant scaffold material.

Permanent therapeutically placed implants often used in situations when regeneration or transplantation are not practical or possible. They include metallic grafts for osteosynthesis, bulk metallic glasses, ceramics, and non-resorbable polymers providing mechanical support. Repair of the tissues on micro scale can also benefit from the biocompatible permanent implants. Vascular graft engineering and repairs of the spinal cord and peripheral nerves are among the most demanding application. Carbon fibers (CF) have superior mechanical and chemical properties, however, their long-time safety was never systematically estimated. The biggest concern comes from residual polymers used for pyrolysis and epoxy laminating resins. Here we attempted to investigate survival of the cells cultured on carbon fibers and to evaluate the tissue responses towards the long-term implanted material. Immortalized rat Schwann cells displayed efficient sporadic attachment to the carbon fibers with survival rate over 90%. Carbon fiber implants in adipose and on connective tissues were tolerable by animals during about 40% of their lifespan with no signs of inflammation on physiological, morphological or gene expression level.

Donation of neural stem cells? Post mortal viability of spinal cord neuronal cells.

Transplantation of cells into central nervous system (CNS) shows a potential for treatment of post-traumatic and neurodegenerative diseases. Cadaver-derived neural cells can help reducing deficit of allogeneic material ready for transplantation. In this study we analyze post-mortal survival of spinal cord neural cells. Maximal time when alive neuronal cells can be recovered form spinal cord of the animals was determined as 56hr for human-size animal and 18hr for rat. Cells with surface expression of ganglioside GD2 and antigen CD24 constituted up to one percent of all recovered alive cells in earlier samples with time dependent decline in percentage. GD2-positive cells from rat spinal cord demonstrated spontaneous and induced electrical activity, which reduces with time post mortem.

Bio-active polymer implants to adipose tissue as in situ source of reprogramming cells.

The implants from natural polymer chitosan was developed by sorption of non-viral DNA vector. These assemblies were shown to be biocompatible and able to transfect the surrounding cells with reprogramming factors. Transgenic rats from the strain allowing visualization of Nanog-positive stem cells received these implants into their adipose deposits. After 8 days we were able to detect in situ-transfected cells. Transfected cells were positive for YFP fluorescence and transiently expressed the markers correlating with stemness.

Peptide-functionalized chitosan-DNA nanoparticles for cellular targeting.

Chitosan-pDNA nanoparticles with various weight ratios (chitosan:pDNA 1:4-8:1) were characterized for particle size, zeta potential, morphology, and pDNA binding efficiency. For targeted gene delivery applications, nanoparticles were functionalized by coupling fluorescent dye and tyrosine kinase receptor B (TrkB) binding peptides on the particle surface. The targetability of the peptide-functionalized nanoparticles was demonstrated in TrkB positive murine transformed monocyte/macrophage cells (RAW 264). It was observed that weight ratio influenced DNA condensation and nanoparticle properties. An increase in the weight ratio decreased the average particle size, but increased the zeta potential. Cell culture studies showed that TrkB-peptide-functionalized nanoparticles bound to cells more effectively than nanoparticles functionalized with a control peptide. The length of the PEG spacer arm of the amine-to-sulfhydryl crosslinker used in the functionalization was found to positively correlate with the cellular attachment efficiency. This study suggests that the peptide-functionalization could be used to target chitosan-pDNA nanoparticles to specific cells.

Modeling reveals that dynamic regulation of c-FLIP levels determines cell-to-cell distribution of CD95-mediated apoptosis.

The expression levels of caspase-8 inhibitory c-FLIP proteins play an important role in regulating death receptor-mediated apoptosis, as their concentration at the moment when the death-inducing signaling complex (DISC) is formed determines the outcome of the DISC signal. Experimental studies have shown that c-FLIP proteins are subject to dynamic turnover and that their stability and expression levels can be rapidly altered. Even though the influence of c-FLIP on the apoptotic behavior of a single cell has been captured in mathematical simulation studies, the effect of c-FLIP turnover and stability has not been investigated. In this study, a mathematical model of apoptosis was developed to analyze how the dynamic turnover and stability of the c-FLIP isoforms regulate apoptotic signaling for both individual cells and cell populations. Intercellular parameter and concentration distributions were used to describe the behavior of cell populations. Monte-Carlo simulations of cell populations showed that c-FLIP turnover is a key determinant of death receptor responses. The fact that the developed model simulates the state of whole cell populations makes it possible to validate it by comparison with empirical data. The proposed modeling approach can be used to further determine limiting factors in the DISC signaling process.

Phosphopeptide enrichment with stable spatial coordination on a titanium dioxide coated glass slide.

Recent advances in phosphoproteomics have established powerful tools to analyze phosphorylation events. However, their spatial localization is lost due to sample homogenization procedures prior to the analysis. Imaging mass spectrometry (IMS) has emerged as a method to visualize the spatial distribution of molecules in tissue samples, but its application is still limited to relatively abundant molecules. Due to low phosphorylation stoichiometry, direct detection and imaging of protein phosphorylation by MS has not been achieved yet. Therefore we have developed a novel phosphopeptide enrichment strategy as a potential tool for in situ affinity imaging MS (AIMS). A specific type of titanium dioxide (TiO2)-coated glass slides was designed and validated with casein tryptic digests for their ability to selectively retain phosphopeptides while maintaining their spatial coordination.

Opposing roles for caspase and calpain death proteases in L-glutamate-induced oxidative neurotoxicity.

Oxidative glutamate toxicity in HT22 murine hippocampal cells is a model for neuronal death by oxidative stress. We have investigated the role of proteases in HT22 cell oxidative glutamate toxicity. L-glutamate-induced toxicity was characterized by cell and nuclear shrinkage and chromatin condensation, yet occurred in the absence of either DNA fragmentation or mitochondrial cytochrome c release. Pretreatment with the selective caspase inhibitors either benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (pan-caspase), N-acetyl-Leu-Glu-His-Asp-aldehyde (caspase 9) or N-acetyl-Ile-Glu-Thr-Asp-aldehyde (caspase 8), significantly increased L-glutamate-induced cell death with a corresponding increase in observed nuclear shrinkage and chromatin condensation. This enhancement of glutamate toxicity correlated with an increase in L-glutamate-dependent production of reactive oxygen species (ROS) as a result of caspase inhibition. Pretreating the cells with N-acetyl-L-cysteine prevented ROS production, cell shrinkage and cell death from L-glutamate as well as that associated with the presence of the pan-caspase inhibitor. In contrast, the caspase-3/-7 inhibitor N-acetyl-Asp-Glu-Val-Asp aldehyde was without significant effect. However, pretreating the cells with the calpain inhibitor N-acetyl-Leu-Leu-Nle-CHO, but not the cathepsin B inhibitor CA-074, prevented cell death. The cytotoxic role of calpains was confirmed further by: 1) cytotoxic dependency on intracellular Ca(2+) increase, 2) increased cleavage of the calpain substrate Suc-Leu-Leu-Val-Tyr-AMC and 3) immunoblot detection of the calpain-selective 145 kDa alpha-fodrin cleavage fragment. We conclude that oxidative L-glutamate toxicity in HT22 cells is mediated via calpain activation, whereas inhibition of caspases-8 and -9 may exacerbate L-glutamate-induced oxidative neuronal damage through increased oxidative stress.

CD73 participates in cellular multiresistance program and protects against TRAIL-induced apoptosis.

The molecular mechanisms underlying the multiresistant phenotype of leukemic and other cancer cells are incompletely understood. We used expression arrays to reveal differences in the gene expression profiles of an apoptosis-resistant T cell leukemia clone (A4) and normally apoptosis-sensitive parental Jurkat cells. CD73 (ecto-5'-nucleotidase) was the most up-regulated gene in the resistant A4 cell clone. A4 cells displayed CD73 surface expression and significant ecto-5'-nucleotidase activity. The role of CD73 was confirmed by transfection of wild-type CD73 into native Jurkat cells, which led to specific resistance against TRAIL-induced apoptosis, but not other types of apoptosis. The protective role of CD73 was further confirmed by small interfering RNA-mediated down-regulation of CD73, restoring TRAIL sensitivity. CD73-mediated resistance was independent of enzymatic activity of CD73, but was reliant on the anchoring of the protein to the membrane via GPI. We suggest that the inhibition of TRAIL signaling works through interaction of CD73 with death receptor 5, as CD73 and death receptor 5 could be coimmunoprecipitated and were shown to be colocalized in the plasma membrane by confocal microscopy. We propose that CD73 is a component of multiresistance machinery, the transcription of which is activated under selective pressure of the immune system.

The detection of micromolar pericellular ATP pool on lymphocyte surface by using lymphoid ecto-adenylate kinase as intrinsic ATP sensor.

Current models of extracellular ATP turnover include transient release of nanomolar ATP concentrations, triggering of signaling events, and subsequent ectoenzymatic inactivation. Given the high substrate specificity for adenylate kinase for reversible reaction (ATP + AMP <--> 2ADP), we exploited lymphoid ecto-adenylate kinase as an intrinsic probe for accurate sensing pericellular ATP. Incubation of leukemic T- and B-lymphocytes with [3H]AMP or [alpha-32P]AMP induces partial nucleotide conversion into high-energy phosphoryls. This "intrinsic" AMP phosphorylation occurs in time- and concentration-dependent fashions via nonlytic supply of endogenous gamma-phosphate-donating ATP, remains relatively resistant to bulk extracellular ATP scavenging by apyrase, and is diminished after lymphocyte pretreatment with membrane-modifying agents. This enzyme-coupled approach, together with confocal imaging of quinacrine-labeled ATP stores, suggests that, along with predominant ATP accumulation within cytoplasmic granules, micromolar ATP concentrations are constitutively retained on lymphoid surface without convection into bulk milieu. High basal levels of inositol phosphates in the cells transfected with ATP-selective human P2Y2-receptor further demonstrate that lymphocyte-surrounding ATP is sufficient for triggering purinergic responses both in autocrine and paracrine fashions. The ability of nonstimulated lymphocytes to maintain micromolar ATP halo might represent a novel route initiating signaling cascades within immunological synapses and facilitating leukocyte trafficking between the blood and tissues.

Live cell detection of caspase-3 activation by a Discosoma-red-fluorescent-protein-based fluorescence resonance energy transfer construct.

A probe consisting of Discosoma red fluorescent protein (DsRed) and enhanced yellow fluorescent protein (EYFP) linked by a 19-amino-acid chain containing the caspase-3 cleavage site Asp-Glu-Val-Asp was developed to monitor caspase-3 activation in living cells. The expression of the tandem construct in mammalian cells yielded a strong red fluorescence when excited with 450- to 490-nm light or with a 488-nm argon ion laser line as a result of fluorescence resonance energy transfer (FRET) from donor EYFP to acceptor DsRed. The advantage over previous constructs using cyan fluorescent protein is that our construct can be used when excitation wavelengths lower than 488nm are not available. To validate the construct, murine HT-22 hippocampal neuronal cells were triggered to undergo CD95-induced neuronal death. An increase in caspase-3 activity was demonstrated by a reduction of FRET in cells transfected with the construct. This was manifested by a dequenching of EYFP fluorescence leading to an increase in EYFP emission and a corresponding decrease in DsRed fluorescence, which correlated with an increase in pro-caspase-3 processing. We conclude that CD95-induced caspase-3 activation in HT-22 cells was readily detected at the single-cell level using the DsRed-EYFP-based FRET construct, making this a useful technology to monitor caspase-3 activity in living cells.

Type-2A protein phosphatase activity is required to maintain death receptor responsiveness.

Type-2A protein phosphatase (PP2A) is a key regulator in many different cell signaling pathways and an important determinant in tumorigenesis. One of the signaling targets of PP2A is the mitogen-activated protein kinase (MAPK/ERK) cascade. In this study, we wanted to determine whether PP2A could be involved in regulation of death receptor activity through its capacity to regulate MAPK/ERK. To this end, we studied the effects of two different routes of protein phosphatase inhibition on death receptor-mediated apoptosis. We demonstrated that the apoptosis mediated by Fas, TNF-alpha, and TRAIL in U937 cells is suppressed by calyculin A, an inhibitor of type-1 and type-2A protein phosphatases. The inhibition of the protein phosphatase activity was shown to subsequently increase the MAPK activity in these cells, and the level of activation corresponded to the degree of suppression of cytokine-mediated apoptosis. A more physiological inhibitor, the intracellular PP2A inhibitor protein I2(PP2A), protected transfected HeLa cells in a similar way from Fas-mediated apoptosis and induced activation of MAPK in I2(PP2A) transfected cells. A corresponding inhibition could also be obtained by stable transfection with a constitutively active form of the MAPK kinase, MKK1 (also referred to as MEK1). The inhibitor-mediated protection was highly efficient in preventing early stages of apoptosis, as no caspase-8 cleavage occurred in these cells. The observed apoptosis suppression is likely to facilitate the tumor-promoting effect of a range of different type-2A protein phosphatase inhibitors, and could explain the reported tumor association of I2(PP2A).

Cdk5 regulates the organization of Nestin and its association with p35.

The intermediate filament protein nestin is characterized by its specific expression during the development of neuronal and myogenic tissues. We identify nestin as a novel in vivo target for cdk5 and p35 kinase, a critical signaling determinant in development. Two cdk5-specific phosphorylation sites on nestin, Thr-1495 and Thr-316, were established, the latter of which was used as a marker for cdk5-specific phosphorylation in vivo. Ectopic expression of cdk5 and p35 in central nervous system progenitor cells and in myogenic precursor cells induced elevated phosphorylation and reorganization of nestin. The kinetics of nestin expression corresponded to elevated expression and activation of cdk5 during differentiation of myoblast cell cultures and during regeneration of skeletal muscle. In the myoblasts, a disassembly-linked phosphorylation of Thr-316 indicated active phosphorylation of nestin by cdk5. Moreover, cdk5 occurred in physical association with nestin. Inhibition of cdk5 activity-either by transfection with dominant-negative cdk5 or by using a specific cdk5 inhibitor-blocked myoblast differentiation and phosphorylation of nestin at Thr-316, and this inhibition markedly disturbed the organization of nestin. Interestingly, the interaction between p35, the cdk5 activator, and nestin appeared to be regulated by cdk5. In differentiating myoblasts, p35 was not complexed with nestin phosphorylated at Thr-316, and inhibition of cdk5 activity during differentiation induced a marked association of p35 with nestin. These results demonstrate that there is a continuous turnover of cdk5 and p35 activity on a scaffold formed by nestin. This association is likely to affect the organization and operation of both cdk5 and nestin during development.