Comparative cellular and molecular analyses of pooled bone marrow multipotent mesenchymal stromal cells during continuous passaging and after successive cryopreservation

The clinical application of human bone marrow derived multipotent mesenchymal stromal cells (MSC) requires expansion, cryopreservation, and transportation from the laboratory to the site of cell implantation. The cryopreservation and thawing process of MSCs may have important effects on the viability, growth characteristics and functionality of these cells both in vitro and in vivo. More importantly, MSCs after two rounds of cryopreservation have not been as well characterized as fresh MSCs from the transplantation perspective. The objective of this study was to determine if the effect of successive cryopreservation of pooled MSCs during the exponential growth phase could impair their morphology, phenotype, gene expression, and differentiation capabilities. MSCs cryopreserved at passage 3 (cell bank) were thawed and expanded up to passage 4 and cryopreserved for the second time. These cells (passive) were then thawed and cultured up to passage 6, and, at each passage MSCs were characterized. As control, pooled passage 3 cells (active) after one round of cryopreservation were taken all the way to passage 6 without cryopreservation. We determined the growth rate of MSCs for both culture conditions in terms of population doubling number (PDN) and population doubling time (PDT). Gene expression profiles for pluripotency markers and tissue specific markers corresponding to neuroectoderm, mesoderm and endoderm lineages were also analyzed for active and passive cultures of MSC. The results show that in both culture conditions, MSCs exhibited similar growth properties, phenotypes and gene expression patterns as well as similar differentiation potential to osteo‐, chondro‐, and adipo‐lineages in vitro. To conclude, it appears that successive or multiple rounds of cryopreservation of MSCs did not alter the fundamental characteristics of these cells and may be used for clinical therapy. J. Cell. Biochem. 113: 3153–3164, 2012. © 2012 Wiley Periodicals, Inc.     

Regulation of cell cycle and stress responses under nitrosative stress in Schizosaccharomyces pombe

Nitric oxide (NO) acts as a signaling molecule in numerous physiological processes but excess production generates nitrosative stress in cells. The exact protective mechanism used by cells to combat nitrosative stress is unclear. In this study, the fission yeast Schizosaccharomyces pombe has been used as a model system to explore cell cycle regulation and stress responses under nitrosative stress. Exposure to an NO donor results in mitotic delay in cells through G2/M checkpoint activation and initiates rereplication. Western blot analysis of phosphorylated Cdc2 revealed that the G2/M block in the cell cycle was due to retention of its inactive phosphorylated form. Interestingly, nitrosative stress results in inactivation of Cdc25 through S-nitrosylation that actually leads to cell cycle delay. From differential display analysis, we identified plo1, spn4, and rga5, three cell cycle-related genes found to be differentially expressed under nitrosative stress. Exposure to nitrosative stress also results in abnormal septation and cytokinesis in S. pombe. In summary we propose a novel molecular mechanism of cell cycle control under nitrosative stress based on our experimental results and bioinformatics analysis.     

New insights into the role of conserved, essential residues in the GTP binding/GTP hydrolytic cycle of large G proteins

The GTP hydrolytic (GTPase) reaction terminates signaling by both large (heterotrimeric) and small (Ras-related) GTP-binding proteins (G proteins). Two residues that are necessary for GTPase activity are an arginine (often called the "arginine finger") found either in the Switch I domains of the alpha subunits of large G proteins or contributed by the GTPase-activating proteins of small G proteins, and a glutamine that is highly conserved in the Switch II domains of Galpha subunits and small G proteins. However, questions still exist regarding the mechanism of the GTPase reaction and the exact role played by the Switch II glutamine. Here, we have characterized the GTP binding and GTPase activities of mutants in which the essential arginine or glutamine residue has been changed within the background of a Galpha chimera (designated alpha(T)*), comprised mainly of the alpha subunit of retinal transducin (alpha(T)) and the Switch III region from the alpha subunit of G(i1). As expected, both the alpha(T)*(R174C) and alpha(T)*(Q200L) mutants exhibited severely compromised GTPase activity. Neither mutant was capable of responding to aluminum fluoride when monitoring changes in the fluorescence of Trp-207 in Switch II, although both stimulated effector activity in the absence of rhodopsin and Gbetagamma. Surprisingly, each mutant also showed some capability for being activated by rhodopsin and Gbetagamma to undergo GDP-[(35)S]GTPgammaS exchange. The ability of the mutants to couple to rhodopsin was not consistent with the assumption that they contained only bound GTP, prompting us to examine their nucleotide-bound states following their expression and purification from Escherichia coli. Indeed, both mutants contained bound GDP as well as GTP, with 35-45% of each mutant being isolated as GDP-P(i) complexes. Overall, these findings suggest that the R174C and Q200L mutations reveal Galpha subunit states that occur subsequent to GTP hydrolysis but are still capable of fully stimulating effector activity.     

Intracellular transport dynamics of endosomes containing DNA polyplexes along the microtubule network

We have explored the transport of DNA polyplexes enclosed in endosomes within the cellular environment by multiple particle tracking (MPT). The polyplex-loaded endosomes demonstrate enhanced diffusion at short timescales (t<7 s) with their mean-square displacement (MSD) Deltax(t)2 scaling as t1.25. For longer time intervals they exhibit subdiffusive transport and have an MSD scaling as t0.7. This crossover from an enhanced diffusion to a subdiffusive regime can be explained by considering the action of motor proteins that actively transport these endosomes along the cellular microtubule network and the thermal bending modes of the microtubule network itself.     

Drosophila homologue of Eps15 is essential for synaptic vesicle recycling

The mammalian protein Eps15 is phosphorylated by EGF receptor tyrosine kinase and has been shown to interact with several components of the endocytic machinery. We have identified a hypomorphic Eps15 mutant in Drosophila which shows reversible paralysis and an altered physiology at restrictive temperatures. In addition, the temperature-sensitive paralytic defect of shibire mutant is enhanced by this mutant. Eps15 is enriched in the larval neuromuscular junction in endocytic 'hot spots' in a pattern similar to Dynamin. Eps15 mutants show a decrease in the alpha-Adaptin levels at the larval neuromuscular junction synapse. Genetic and biochemical studies of interactions with components of the endocytic machinery suggest that Eps15 has an important role in synaptic vesicle recycling and regulates recruitment of alpha-Adaptin.     

55 kDa outer-membrane protein from short-chain fatty acids exposed Salmonella enterica serovar Typhi induces apoptosis in macrophages

Various pathogens including Salmonella species are known to induce apoptosis in host cell types during their infection processes. However, the bacterial components capable of inducing apoptosis have not been fully understood. It is now known that in vivo expression of virulence determinants differ from the expression under in vitro conditions. Therefore, in the present study, attempts were made to evaluate the apoptotic potential of outer-membrane protein (OMP) from short-chain fatty acids (SCFA) exposed Salmonella enterica serovar Typhi. Short-chain fatty acids exposure is one of the in vivo stresses encountered by the pathogen in the intestine. Therefore, to simulate the in vivo condition, S. enterica serovar Typhi was grown in the presence of SCFA and its OMP profile was analyzed. The apoptotic potential of 55 kDa protein expressed with enhanced intensity under the SCFA stress was evaluated. Murine peritoneal macrophages interacted with 55 kDa protein showed DNA fragmentation, changes in fluorescence and exposure of phosphatidylserine on their outer leaflets. Levels of nitrite and citrulline were found to be increased in the supernatant of macrophages after interacting them with 55 kDa protein. However, the enzymatic activity of superoxide dismutase was found to be decreased as compared to that of the control (uninteracted) macrophages. These observations indicate that increased levels of nitrite and decreased levels of superoxide dismutase may be one of the mechanisms to induce apoptosis in macrophages by SCFA induced 55 kDa OMP. These findings may help us better understand the pathophysiology of the disease during the host pathogen interactions.     

Tumour necrosis factor alpha mediated apoptosis in murine macrophages by Salmonella enterica serovar Typhi under oxidative stress

Invasive Salmonella has been reported to induce apoptosis of macrophages as part of its infection process, which may allow it to avoid detection by the innate immune system. However, the induction of apoptosis under the different host environments remains to be examined, including the oxidative stress experienced by pathogens in the macrophage milieu. To simulate in vivo oxidative conditions, Salmonella enterica serovar Typhi was grown in the presence of hydrogen peroxide and its ability to induce apoptosis of murine macrophages was assessed. Analysis of data revealed that oxidative stressed S. Typhi caused apoptotic cell death in 51% of macrophages, whereas S. Typhi grown under normal conditions accounted for apoptotic cell death in only 32% of macrophages. A significant increase in the levels of oxidants and decrease in the antioxidant was also observed which correlated with the increased generation of tumour necrosis factor alpha, interleukin-1alpha and interleukin-6. These results suggest that tumour necrosis factor alpha in conjunction with other cytokines may induce apoptotic cell death through the up-regulation of lipid peroxidation and down-regulation of superoxide dismutase. This finding may help us to understand better the host-pathogen interactions and may be of clinical importance in the development of preventive intervention against infection.     

Emission of methane and carbon dioxide and earthworm survival during composting of pharmaceutical sludge and spent mycelia

Emissions of methane (CH4) and carbon dioxide (CO2) from spent mycelia of the mold Penicilium notatum and sludge from the effluent treatment facility (ETPS) of a pharmaceutical industry were estimated twice during a two-week composting before vermicomposting. These wastes are dumped in landfills or sometimes used in agricultural fields and no reports are available on their greenhouse gas producing potentials. The solid wastes contained appreciable organic carbon and nitrogen while very high Fe, Mn and Zn were found in ETPS only. Pure wastes did not support germination of Vigna radiata L. while mixing soil with ETPS and spent mycelia at the ratios of 12:1 and 14:1 led to 80% and 50% germination, respectively. The wastes were mixed with cowdung at the ratios of 1:1, 1:3 and 3:1 for composting. Carbon dioxide emissions were always significantly higher than CH4 emissions from all the treatments due to prevalence of aerobic condition during composting. From some treatments, CH4 emissions increased with time, indicating increasing activity of anaerobic bacteria in the waste mixtures. Methane emissions ranged from 21.6 to 231.7 microg m(-2) day(-1) while CO2 emissions were greater than thousand times at 39.8-894.8 mg m(-2) day(-1). The amount of C emitted as CH4-C and CO2-C from ranged from 0.007% to 0.081% of total C composted. Cowdung emitted highest CH4 followed by spent mycelia and ETPS while ETPS emitted more CO2 than spent mycelia but lesser than cowdung. Global warming potential of emitted CH4 was found to be in the range of 10.6-27.7 mg-CO2-equivalent on a 20-year time horizon. The results suggest that pharmaceutical wastes can be an important source of CH4 and CO2 during composting or any other stockpiling under suitable moisture conditions. The waste mixtures were found not suitable for vermicomposting after two weeks composting and earthworms did not survive long in the mixtures.     

Schlafen-3: A novel regulator of intestinal differentiation

Schlafen-3 (Slfn-3), a novel gene, has been shown to be a negative regulator of proliferation. The current investigation was undertaken to determine whether Slfn-3 might play a role in regulating cellular differentiation. Butyric acid, a short chain fatty acid, which induced differentiation of intestinal cells as evidenced by increased alkaline phosphatase (ALP) activity in the rat small intestinal IEC-6 cells, also produced a marked increase in Slfn-3 expression. Furthermore, overexpression of Slfn-3 caused stimulation of ALP activity in IEC-6 cells, which was exacerbated by butyrate. On the other hand, downregulation of Slfn-3 by slfn-3-si-RNA greatly attenuated the butyrate-mediated induction of differentiation of IEC-6 cells. Additionally, we observed that increased expression of Slfn-3 in colon cancer HCT-116 cells stimulated TGF-β expression and modulated expression of its downstream effectors as evidenced by increased expression of p27kip1 and downregulation of CDK-2. In addition, Slfn-3 increases E-cadherin expression but downregulates β-catenin. In conclusion, our data show that Slfn-3 plays a critical role in regulating intestinal mucosal differentiation. Furthermore our data also show that TGF-β signaling pathway plays an important role in mediating slfn-3 induced differentiation.     

Association of the functional KL-VS variant of Klotho gene with early-onset ischemic stroke

Genetic variants of Klotho have been reported to be associated with human longevity and atherosclerotic vascular events and risk factors. However, very few studies have explored their association with ischemic stroke. We hypothesized that the functional KL-VS and the exonic C1818T variants of Klotho gene may be associated with ischemic stroke in Indian population. We enrolled a total of 460 patients with ischemic stroke and 574 age- and gender-matched controls for the study. Genotyping was done by polymerase chain reaction and restriction fragment length polymorphism. Contrary to other Asian reports, KL-VS variant was polymorphic in our population, with a frequency distribution similar to that of Caucasians. The frequency distribution of the C1818T variant was similar to previously reports in Asians. A differential effect of age on association of Klotho KL-VS variant with ischemic stroke was observed. In subjects aged ≤40 years, the KL-VS homozygotes, 352FF and 352VV, had ~1.5-fold (OR=1.57; 95% CI: 1.02-2.40, p=0.038) and ~3-fold (OR=3.29; 95%CI: 1.02-10.56, p=0.046) higher risk of stroke compared to heterozygotes, whereas in the older group (aged >40 years) no significant association was observed. The C1818T variant was not associated with ischemic stroke. We conclude that KL-VS homozygosity could contribute to early-onset stroke in India. Larger studies in other ethnic populations are warranted to determine the role of these gene variants in the etiology of stroke occurring in the young.