Genes differentially expressed by methylprednisolone in vivo in CD4 T lymphocytes from multiple sclerosis patients: potential biomarkers.

Intravenous methylprednisolone (IVMP) is the gold standard treatment in acute relapses of multiple sclerosis. Knowing the response to IVMP in advance could facilitate earlier selection of patients for subsequent courses of therapy. However, molecular mechanisms and changes in gene expression induced by methylprednisolone remain unknown. The aim of the study was to identify in vivo differentially expressed genes in relapsing-remitting multiple sclerosis patients after 3-6 days of treatment with IVMP. For this purpose, whole-genome transcription profiling of CD4+ T lymphocytes was performed before and after treatment with IVMP in 8 relapsing-remitting multiple sclerosis patients during relapse using Human GE 4x44K v2 microarrays. Differentially expressed genes were identified using a paired t test on GeneSpring v13.0 software. A P-value <0.001 and a twofold change were considered significant. Microarray data were confirmed using real-time PCR. Microarray revealed changes in gene expression: four genes were downregulated (B3GNT3, ZNF683, IFNG and TNF) and seven upregulated (DEFA4, CTSG, DEFA8P, AZU1, MPO, ELANE and PRTN3). Pathway analysis revealed the transforming growth factor-ß signaling pathway to be affected. Comparison with previously published data on in vitro methylprednisolone-regulated genes showed that SMAD7, TNF and CHI3L1 were also downregulated in vivo in relapsing-remitting multiple sclerosis patients. In summary, we performed the first in vivo transcriptome analysis in CD4+ T lymphocytes before and after the treatment with IVMP in patients with multiple sclerosis. Identification of differentially expressed genes in patients receiving IVMP could improve our understanding of the molecular mechanisms underlying the therapeutic effects of IVMP and highlight potential biomarkers of the response to IVMP.

Human mesenchymal stem cell-replicative senescence and oxidative stress are closely linked to aneuploidy.

In most clinical trials, human mesenchymal stem cells (hMSCs) are expanded in vitro before implantation. The genetic stability of human stem cells is critical for their clinical use. However, the relationship between stem-cell expansion and genetic stability is poorly understood. Here, we demonstrate that within the normal expansion period, hMSC cultures show a high percentage of aneuploid cells that progressively increases until senescence. Despite this accumulation, we show that in a heterogeneous culture the senescence-prone hMSC subpopulation has a lower proliferation potential and a higher incidence of aneuploidy than the non-senescent subpopulation. We further show that senescence is linked to a novel transcriptional signature that includes a set of genes implicated in ploidy control. Overexpression of the telomerase catalytic subunit (human telomerase reverse transcriptase, hTERT) inhibited senescence, markedly reducing the levels of aneuploidy and preventing the dysregulation of ploidy-controlling genes. hMSC-replicative senescence was accompanied by an increase in oxygen consumption rate (OCR) and oxidative stress, but in long-term cultures that overexpress hTERT, these parameters were maintained at basal levels, comparable to unmodified hMSCs at initial passages. We therefore propose that hTERT contributes to genetic stability through its classical telomere maintenance function and also by reducing the levels of oxidative stress, possibly, by controlling mitochondrial physiology. Finally, we propose that aneuploidy is a relevant factor in the induction of senescence and should be assessed in hMSCs before their clinical use.

Culture of human mesenchymal stem cells at low oxygen tension improves growth and genetic stability by activating glycolysis.

Expansion of human stem cells before cell therapy is typically performed at 20% O(2). Growth in these pro-oxidative conditions can lead to oxidative stress and genetic instability. Here, we demonstrate that culture of human mesenchymal stem cells at lower, physiological O(2) concentrations significantly increases lifespan, limiting oxidative stress, DNA damage, telomere shortening and chromosomal aberrations. Our gene expression and bioenergetic data strongly suggest that growth at reduced oxygen tensions favors a natural metabolic state of increased glycolysis and reduced oxidative phosphorylation. We propose that this balance is disturbed at 20% O(2), resulting in abnormally increased levels of oxidative stress. These observations indicate that bioenergetic pathways are intertwined with the control of lifespan and decisively influence the genetic stability of human primary stem cells. We conclude that stem cells for human therapy should be grown under low oxygen conditions to increase biosafety.

Comparative analysis of Drosophila melanogaster and Caenorhabditis elegans gene expression experiments in the European Soyuz flights to the International Space Station.

The European Soyuz missions have been one of the main routes for conducting scientific experiments onboard the International Space Station, which is currently in the construction phase. A relatively large number of life and physical sciences experiments as well as technology demonstrations have been carried out during these missions. Included among these experiments are the Gene experiment during the Spanish "Cervantes" Soyuz mission and the ICE-1st experiment during the Dutch "Delta" mission. In both experiments, full genome microarray analyses were carried out on RNA extracted from whole animals recovered from the flight. These experiments indicated relatively large scale changes in gene expression levels in response to spaceflight for two popular model systems, Drosophila melanogaster (Gene) and Caenorabditis elegans (ICE-1st). Here we report a comparative analysis of results from these two experiments. Finding orthologous genes between the fruit fly and the nematode was far from straightforward, reducing the number of genes that we could compare to roughly 20% of the full comparative genome. Within this sub-set of the data (2286 genes), only six genes were found to display identical changes between species (decreased) while 1809 genes displayed no change in either species. Future experiments using ground simulation techniques will allow producing a better, more comprehensive picture of the putative set of genes affected in multicellular organisms by changes in gravity and getting a deeper understanding of how animals respond and adapt to spaceflight.

Modulation of life-span by histone deacetylase genes in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae has a limited life-span, which is measured by the number of divisions that individual cells complete. Among the many changes that occur as yeasts age are alterations in chromatin-dependent transcriptional silencing. We have genetically manipulated histone deacetylases to modify chromatin, and we have examined the effect on yeast longevity. Deletion of the histone deacetylase gene RPD3 extended life-span. Its effects on chromatin functional state were evidenced by enhanced silencing at the three known heterochromatic regions of the genome, the silent mating type (HM), subtelomeric, and rDNA loci, which occurred even in the absence of SIR3. Similarly, the effect of the rpd3Delta on life-span did not depend on an intact Sir silencing complex. In fact, deletion of SIR3 itself had little effect on life-span, although it markedly accelerated the increase in cell generation time that is observed during yeast aging. Deletion of HDA1, another histone deacetylase gene, did not result in life-span extension, unless it was combined with deletion of SIR3. The hda1Delta sir3Delta resulted in an increase in silencing, but only at the rDNA locus. Deletion of RPD3 suppressed the loss of silencing in rDNA in a sir2 mutant; however, the silencing did not reach the level found in the rpd3Delta single mutant, and RPD3 deletion did not overcome the life-span shortening seen in the sir2 mutant. Deletion of both RPD3 and HDA1 caused a decrease in life-span, which resulted from a substantial increase in initial mortality of the population. The expression of both of these genes declines with age, providing one possible explanation for the increase in mortality during the life-span. Our results are consistent with the loss of rDNA silencing leading to aging in yeast. The functions of RPD3 and HDA1 do not overlap entirely. RPD3 exerts its effect on chromatin at additional sites in the genome, raising the possibility that events at loci other than rDNA play a role in the aging process.

The role of gravity in the evolutionary emergence of multicellular complexity: microgravity effects on arthropod development and aging.

While experiments carried out in Space with isolated cells have shown that eucaryotic cells are able to sense and respond to the absence of gravity by modifying their reactions, experiments in which more complex processes have been investigated, such as Biological Systems undergoing development under Microgravity, have been surprisingly unaffected by the space environment. This can be considered a curious result since all organisms are evolutionarily adapted to the current level of the gravity force in our planet and should eventually change if this parameter will vary in a permanent manner. In fact, the small effects of the modifications in gravity on development in short term experiments may be equivalent to the difficulties in detecting the involvement of other basic physical processes such as diffusion-controled auto-organizative reactions in currently developing biological systems. An apparent exception to this lack of effect is experiments where brine shrimp dormant gastrulae directly exposed to the space environment accumulate developmental defects as a consequence of cosmic irradiation. In this article we discuss the idea that at a certain stage during the evolutionary emergence of multicellular organisms the cues laid by generic forces such as gravity were involved in the evolutionary organization of these primitive organisms. As evolution proceed, these early mechanisms may have been obscured and/or made redundant by the appearance of new internal, environment-independent biological regulatory mechanisms. On the other hand, behavioral responses that may be important, for example, in setting the life-spans of organisms may still be more readily susceptible to manipulation by external cues as experiments carried out by our group in Space and on the ground with Drosophila melanogaster indicate. Grant Numbers: ESP-1775.

Epigenetic stratification: the role of individual change in the biological aging process.

Aging is a complex process. It consists of a diverse assortment of seemingly random manifestations that occur in the individual, the mutual relationship and impact on mortality of which is frequently obscure. We derive a simple equation to model the aging process based on scale invariant and increasing change. The solution to this equation indicates that this change itself, irrespective of its quality, is the cause and not simply the effect of aging. This model establishes loss of homeostasis as a fundamental feature of aging. The model is deterministic, but it supports the stochastic nature of age changes. Paradoxically, this model states that a sufficient augmentation of aging processes results in a lack of aging. Experimental evidence in support of this model is presented that spans the levels of population mortality rates, cellular spatial organization, and gene dysregulation.

Effects of the space environment on Drosophila melanogaster development. Implications of the IML-2 experiment.

One hundred and sixty Drosophila females laid several thousands of embryos during the 14.5 days of the IML-2 spaceflight. The progeny were either recovered frozen (embryos at final stages of development and larvae), or maintained alive developing further until adulthood. All embryos, larvae, pupae and imagoes recovered were normal in morphology and function. Results from earlier experiments were reproduced in IML-2 with a better experimental design. We confirm that in Space there is a stimulation of oogenesis and that development is slightly delayed when compared to that of synchronous parallel ground controls. Nevertheless, it is clear from the accompanying 1 x g flight control centrifuge and from the 1.4 x g ground centrifuge samples, that centrifugation itself can produce similar effects, emphasizing the importance of reevaluating the role of the 1 x g on-flight controls. The results emphasize the apparent paradox that simple cellular model systems in microgravity show alterations in many fundamental processes such as those involved in cell signalling, while development, relying heavily on these key cellular mechanisms can proceed quite normally in the absence of gravity. The effects on development are small and more the consequence of a reaction to the abnormal Space environment in general than a specific effect of microgravity.

Microgravity effects on Drosophila melanogaster behavior and aging. Implications of the IML-2 experiment.

Earlier Space experiments had indicated that young male Drosophila flies exposed to microgravity showed an acceleration in aging. In a 14.5-day Space Shuttle Flight we sent 300 young male flies with the purpose of confirming these findings and to establish whether changes in the behavior of the flies were responsible for the effect in accordance with the proposal that alterations in mitochondrial metabolism may be involved in the aging response. By repeatedly video-recording, we have found a very marked increase in the locomotor activity of the fruitflies in Space. The males showed an accelerated aging response upon recovery, both in terms of physiological vitality assays (mating and negative geotaxis) and of life-span curves. The involvement of mitochondrial metabolism is also suggested by the finding of a greater decrease in mitochondrial 16S ribosomal RNA in the microgravity exposed flies than in ground controls. On the other hand, a parallel 1 x g centrifuge control did not show such differences in the life-span curves when compared to flies exposed to a similar centrifugation on the ground. Drosophila females also increased their locomotor activity but did not show differential changes in the life-span curves. These results are discussed in terms of the current mechanisms of aging in multicellular eukaryotic organisms.

Preservation of viable biological samples for experiments in space laboratories.

Standard viable preservation methods for biological samples using low temperatures have been investigated concerning their storage capabilities under higher temperature levels than usual. For a representative set of organism classes (plants, mammalian cells, arthropods and aquatic invertebrates), the minimum appropriate storage conditions have been identified by screening storage temperatures at -196 degrees, -80 degrees, -20 degrees, +4 degrees, +20 degrees/25 degrees C for periods from 2 days to 4 weeks. For storage below 0 degree C, as a typical cryopreservative, dimethylsulfoxide (DMSO) was used. For some samples, the addition of trehalose (as cryopreservative) and the use of a nitrogen atmosphere were investigated. After storage, the material was tested for vitality. The findings demonstrated that acceptable preservation can be achieved under higher storage temperatures than are typically applied. Small, dense cultured plant cells survive for 21 d when moderately cooled (+4 degrees to -20 degrees C); addition of trehalose enhances viability at -20 degrees C. For mammalian cells, the results show that human lymphocytes can be preserved for 3 d at 25 degrees C, 7 d at 4 degrees C and 28 d at -80 degrees C. Friend leukaemia virus transformed cells can be stored for 3 d at 25 degrees C, 14 d at 4 degrees C and 28 d at -80 degrees C. Hybridoma cells can be kept 7 d at 4 degrees C and 28 d at -20 degrees C or -80 degrees C. Model arthropod systems are well preserved for 2 weeks if maintained at lower temperatures that vary depending on the species and/or stage of development; e.g., 12 degrees C for Drosophila imagoes and 4-6 degrees C for Artemia nauplii. For aquatic invertebrates such as sea urchins, embryonic and larval stages can be preserved for several weeks at +6 degrees C, whereas sperm and eggs can best be stored at + 4 degrees C for up to 5 d at maximum. These results enhance the range of feasible space experiments with biological systems. Moreover, for typical terrestrial preservation methods, considerable modification potential is identified.

Phosphorylation of calmodulin by plasma-membrane-associated protein kinase(s).

Plasma-membrane-associated protein kinase(s) from normal rat liver phosphorylates exogenous bovine brain calmodulin in the absence of Ca2+ and in the presence of histone or poly(L-lysine). Maximum levels of calmodulin phosphorylation are obtained at a poly(L-lysine)/calmodulin molar ratio of 0.4. Phosphoamino acid analysis revealed that calmodulin is phosphorylated on serine, threonine and tyrosine residues. Endogenous plasma-membrane-associated calmodulin was also phosphorylated by plasma-membrane-associated protein kinase(s) in the absence of added cationic protein or polypeptide. The identity of endogenous phosphocalmodulin was confirmed by immunoprecipitation with a specific anti-calmodulin monoclonal antibody. Ehrlich ascites tumor cell plasma membranes do not contain endogenous calmodulin. However, membrane-associated protein kinase(s) from these tumor cells phosphorylates bovine brain calmodulin in the presence of poly(L-lysine). These data demonstrate that phosphocalmodulin is present in liver plasma membranes and suggest that this post-translational modification could have a physiological role in this location.

Differential response of the epidermal growth factor receptor tyrosine kinase activity to several plant and mammalian lectins.

Biosignalling via lectins may involve modulation of protein kinase activities. This aspect of the biological action of mammalian and plant lectins has been investigated for their effect on the activity of the isolated epidermal growth factor receptor (EGFR). The constitutive tyrosine kinase activity of the epidermal growth factor receptor from rat liver, isolated by calmodulin-affinity chromatography, was activated by concanvalin A (ConA), and wheat germ agglutinin (WGA) to a similar extent as the measured enhancement induced by EGF. In contrast, two mannose-specific lectins, the mannan-binding protein (MBP) and serum amyloid P component (SAP), isolated from human serum, have inhibitory effects, both in the absence and presence of EGF. The differential effects of these lectins were tested using as phosphorylatable substrates a co-polymer of glutamic acid-tyrosine, as well as calmodulin. However, two galactoside-specific lectins, the laminin-binding beta-galactoside-binding 14 kDa lectin, isolated from bovine heart (14K-BHL), and the alpha/beta-galactoside-binding lectin, isolated from mistletoe (Viscum album L.) leaves (VAA), do not inhibit the EGFR tyrosine kinase activity. The sugar dependence of the lectin-mediated action was studied by inhibition assays. Mannose and a mannose-containing neoglycoprotein prevent the activating effect of ConA, and N-acetyl-D-glucosamine partially prevents the activation produced by WGA. However, mannose and mannose-containing neoglycoprotein were ineffective to reduce the inhibitory effect of MBP or SAP.(ABSTRACT TRUNCATED AT 250 WORDS)

Phosphorylation of calmodulin by the epidermal-growth-factor-receptor tyrosine kinase.

An epidermal-growth-factor(EGF)-receptor preparation isolated by calmodulin-affinity chromatography from rat liver plasma membranes is able to phosphorylate calmodulin. Calmodulin phosphorylation was enhanced 3-8-fold by EGF, was dependent on the presence of a polycation or basic protein and was inhibited by micromolar concentrations of Ca2+. Phosphate incorporation into calmodulin occurs predominantly on tyrosine residues. Partial proteolysis of phosphocalmodulin by thrombin identifies Tyr99, located in the third calcium-binding domain of calmodulin, as the phosphorylated residue. Stoichiometric measurements show a 32P/calmodulin molar ratio of approximately 1 when optimal phosphorylation conditions are used.

Calmodulin inhibits the epidermal growth factor receptor tyrosine kinase.

We demonstrate in this report that the epidermal growth factor (EGF) receptor from rat liver can be isolated by calmodulin affinity chromatography by binding in the presence of Ca2+ and elution with a Ca(2+)-chelating agent. The bulk of the EGF receptor is not eluted by a NaCl gradient in the presence of Ca2+. We ascertained the identity of the isolated receptor by immunoblot and immunoprecipitation using a polyclonal antibody against an EGF receptor from human origin. The purified receptor is autophosphorylated in tyrosine residues in an EGF-stimulated manner, and EGF-dependent phosphorylation of serine residues was also detected. Both the EGF and the transforming growth factor-alpha stimulate the tyrosine-directed protein kinase activity of the isolated receptor with similar affinities. Furthermore, we demonstrate that calmodulin inhibits the EGF-dependent tyrosine-directed protein kinase activity associated to the receptor in a concentration-dependent manner. This inhibition is partially Ca2+ dependent and is not displaced by increasing the concentration of EGF up to an EGF/calmodulin ratio of 10 (mol/mol). In addition, calmodulin was phosphorylated in an EGF-stimulated manner in the presence of a basic protein (histone) as cofactor and in the absence, but not in the presence, of Ca2+.

A novel adenylylation process in liver plasma membrane-bound proteins.

Rat liver plasma membrane contains five distinct polypeptides of apparent molecular mass of 130, 120, 110, 100, and 86 kDa which are labeled upon incubation with [alpha-32P]ATP as well as with [gamma-32P]ATP. Covalently bound adenosine 5'-monophosphate to some of the polypeptides was identified using nonhydrolyzable analogues of ATP. Chase experiments of alpha-32P-nucleotide-labeled polypeptides with different nonradiolabeled phosphocompounds and sensitivity to different inhibitors demonstrate that the 86-kDa polypeptide is a phosphoesterase, forming a catalytic intermediate. On the other hand, the comparative slow rate of turnover of the polypeptides of higher molecular mass (130, 120, 110, and 100 kDa) suggests that the bound AMP could play a regulatory rather than a catalytic role. Using the nonhydrolyzable ATP analogue [alpha, beta-methylene]ATP and dilution experiments with Triton X-100-solubilized membranes, it has been possible to identify the 130-kDa adenylylated polypeptide as a possible target of an adenylylating system. These polypeptides, except the 86-kDa phosphoesterase, are affected in their electrophoretic mobility in the absence of beta-mercaptoethanol. An intercatenary disulfide bond(s) appear(s) to link the polypeptide(s) of 120 kDa and/or 110 kDa in a dimeric structure of apparent molecular mass of 240 kDa. All five polypeptides labeled with [alpha-32P]ATP are glycoproteins bound to the cell plasma membrane.