Detection of GM1-gangliosidosis in newborn dried blood spots by enzyme activity and biomarker assays using tandem mass spectrometry.

GM1-gangliosidosis is a rare autosomal recessive lysosomal storage disease caused by deficiency of ß-galactosidase (GLB1). Newborn screening (NBS) may be warranted in the near future given the initiation of a number of gene therapy clinical trials. Here, we report a tandem mass spectrometry (MS/MS) enzymatic assay of GLB1 using dried blood spots (DBS), and the demonstration that GLB1 activities in newborn DBS from seven GM1-gangliosidosis patients are well below those measured in random newborn DBS. MS/MS analysis of two glycan biomarkers, dp5 and A2G2, shows high elevation in newborn DBS from GM1-gangliosidosis compared to the levels in the nonaffected reference range.

A Single Injection of an Optimized Adeno-Associated Viral Vector into Cerebrospinal Fluid Corrects Neurological Disease in a Murine Model of GM1 Gangliosidosis.

GM1 gangliosidosis is a rare neurodegenerative lysosomal storage disease caused by loss-of-function mutations in the gene encoding beta-galactosidase (ß-gal). There are no approved treatments for GM1 gangliosidosis. Previous studies in animal models have demonstrated that adeno-associated viral (AAV) vector-mediated gene transfer to the brain can restore ß-gal expression and prevent the onset of neurological signs. We developed an optimized AAV vector expressing human ß-gal and evaluated the efficacy of a single intracerebroventricular injection of this vector into the cerebrospinal fluid (CSF) of a murine disease model. The AAV vector administration into the CSF increased ß-gal activity in the brain, reduced neuronal lysosomal storage lesions, prevented the onset of neurological signs and gait abnormalities, and increased survival. These findings demonstrate the potential therapeutic activity of this vector and support its subsequent development for the treatment of GM1 gangliosidosis.

Targeted delivery of nitric oxide via a 'bump-and-hole'-based enzyme-prodrug pair.

The spatiotemporal generation of nitric oxide (NO), a versatile endogenous messenger, is precisely controlled. Despite its therapeutic potential for a wide range of diseases, NO-based therapies are limited clinically due to a lack of effective strategies for precisely delivering NO to a specific site. In the present study, we developed a novel NO delivery system via modification of an enzyme-prodrug pair of galactosidase-galactosyl-NONOate using a 'bump-and-hole' strategy. Precise delivery to targeted tissues was clearly demonstrated by an in vivo near-infrared imaging assay. The therapeutic potential was evaluated in both rat hindlimb ischemia and mouse acute kidney injury models. Targeted delivery of NO clearly enhanced its therapeutic efficacy in tissue repair and function recovery and abolished side effects due to the systemic release of NO. The developed protocol holds broad applicability in the targeted delivery of important gaseous signaling molecules and offers a potent tool for the investigation of relevant molecular mechanisms.

Bottom-up single-molecule strategy for understanding subunit function of tetrameric ß-galactosidase.

In this paper, we report an example of the engineered expression of tetrameric ß-galactosidase (ß-gal) containing varying numbers of active monomers. Specifically, by combining wild-type and single-nucleotide polymorphism plasmids at varying ratios, tetrameric ß-gal was expressed in vitro with one to four active monomers. The kinetics of individual enzyme molecules revealed four distinct populations, corresponding to the number of active monomers in the enzyme. Using single-molecule-level enzyme kinetics, we were able to measure an accurate in vitro mistranslation frequency (5.8 × 10-4 per base). In addition, we studied the kinetics of the mistranslated ß-gal at the single-molecule level.

Functional characterization of persimmon ß-galactosidase gene DkGAL1 in tomato reveals cell wall modification related to fruit ripening and radicle elongation.

Cell wall metabolism during fruit ripening is a highly organized process that involves complex interplay among various cell wall hydrolases. Among these cell wall hydrolases, ß-galactosidase has been identified to participate in cell wall metabolism via its ability to catalyze galactosyl metabolism from the large and complex side chains of cell walls. In this study, the galactose content in the pericarp increased during persimmon fruit ripening, but cell wall galactosyl residues decreased, indicating a relationship between galactose metabolism and persimmon fruit ripening. Expression of a previously isolated ß-galactosidase gene, DkGAL1, increased 25.01-fold during fruit ripening. Heterologous expression of DkGAL1 under the CaMV 35S promoter in tomato accelerated on-plant and postharvest fruits ripening. The fruit firmness of one of transgenic line, OE-18, was 23.83% lower than that of WT at the breaker stage. The transgenic fruits produced more ethylene by promoting the expression of ethylene synthesis-related genes and cell wall degradation-related genes. Overexpression of DkGAL1 in tomato also reduced cell-to-cell adhesion and promoted both wider intercellular spaces and less cell compaction in transgenic fruit structures. Moreover, DkGAL1 was involved in seed germination and radicle elongation in transgenic tomato seeds. These results confirm the role of DkGAL1 in fruit ripening and suggest that this gene alters galactose metabolism in the fruit, which can promote ripening and reduce cellular adhesion. In addition, the role of DkGAL1 is not limited to fruit softening; DkGAL1 was also involved in seed germination and radicle elongation in transgenic tomato seeds.

Diagnostic challenge for the rare lysosomal storage disease: Late infantile GM1 gangliosidosis.

BACKGROUND: GM1 gangliosidosis is a rare lysosomal storage disorder caused by GLB1 mutations. Because of its extreme rarity and symptoms that overlap with other neurodegenerative diseases, its diagnosis is sometimes challenging, especially in the late infantile form with less severe phenotype. We aim to expand the clinical and genetic spectrum of late infantile GM1 gangliosidosis. METHODS: We confirmed a diagnosis of GM1 gangliosidosis based on GLB1 mutations and/or the deficiency of ß-galactosidase activity. We identified the first two cases by whole-exome sequencing, and then the other six cases by direct sequencing of GLB1 with enzyme analysis. RESULTS: All eight patients presented with developmental delay or regression during late infancy and later developed epilepsy, mostly intractable generalized tonic seizures. No clinical signs of storage disorders were noted except for skeletal abnormalities. Interestingly, we found aspartate transaminase (AST) elevations alone with normal alanine transaminase (ALT) levels in all patients. The recurrent mutation, p.D448V in GLB1, accounted for 50.0% of total alleles in our cohort. CONCLUSIONS: With a high index of clinical suspicion, skeletal survey and AST level would be important for early diagnosis of GM1 gangliosidosis. In addition, we would highlight the clinical usefulness of whole-exome sequencing in the diagnosis of non-classical presentation of ultra-rare neurodegenerative disease in children.

Structural and functional analysis of tomato ß-galactosidase 4: insight into the substrate specificity of the fruit softening-related enzyme.

Plant ß-galactosidases hydrolyze cell wall ß-(1,4)-galactans to play important roles in cell wall expansion and degradation, and turnover of signaling molecules, during ripening. Tomato ß-galactosidase 4 (TBG4) is an enzyme responsible for fruit softening through the degradation of ß-(1,4)-galactan in the pericarp cell wall. TBG4 is the only enzyme among TBGs 1-7 that belongs to the ß-galactosidase/exo-ß-(1,4)-galactanase subfamily. The enzyme can hydrolyze a wide range of plant-derived (1,4)- or 4-linked polysaccharides, and shows a strong ability to attack ß-(1,4)-galactan. To gain structural insight into its substrate specificity, we determined crystal structures of TBG4 and its complex with ß-d-galactose. TBG4 comprises a catalytic TIM barrel domain followed by three ß-sandwich domains. Three aromatic residues in the catalytic site that are thought to be important for substrate specificity are conserved in GH35 ß-galactosidases derived from bacteria, fungi and animals; however, the crystal structures of TBG4 revealed that the enzyme has a valine residue (V548) replacing one of the conserved aromatic residues. The V548W mutant of TBG4 showed a roughly sixfold increase in activity towards ß-(1,6)-galactobiose, and ~0.6-fold activity towards ß-(1,4)-galactobiose, compared with wild-type TBG4. Amino acid residues corresponding to V548 of TBG4 thus appear to determine the substrate specificities of plant ß-galactosidases towards ß-1,4 and ß-1,6 linkages.

The activity of three glycosidases (ß-Ν-acetyloglucosaminidase, α-mannosidase, and ß-galactosidase) in the follicular fluid and in the maturation medium affects bovine oocyte maturation.

We studied the role of follicular fluid's (FF) glycosidase (α-mannosidase [α-ΜΑΝ], ß-Ν-acetyloglucosaminidase [NAGASE], ß-galactosidase [ß-GAL]) activity during IVM of bovine oocytes. Oocytes were allocated into two groups according to the follicular size (small follicle [SF]: 2-5 mm, large follicle [LF]: >5-8 mm). In experiment 1, cumulus-oocyte complexes (COCs) quality was evaluated according to morphologic criteria (grades A, B-C, D); oocyte (n = 801) nuclear maturation was assessed after 24 hours of incubation. Bovine embryos were produced in vitro in groups (experiment 2, n = 1503 oocytes) or individually (experiment 3, n = 50 oocytes). More grade-A and -BC COCs were collected from SF and LF groups, respectively (P < 0.05). Maturation rate (experiment 1) and cleavage rate (experiments 2 and 3) were similar in SF and LF groups. Activity of all glycosidases in FF was higher (P < 0.05) in SF group than in LF group, whereas in maturation medium of SF group it was, overall, significantly lower than in that of LF (experiments 2 and 3). In FF of SF group, NAGASE positively associated with grade-A oocytes and negatively with BC oocytes; increased ß-GAL was associated with degenerated oocytes. Cleavage rate in LF group, related negatively to NAGASE and positively to α-MAN in maturation medium. These results indicate that during maturation, COCs release NAGASE and consume ß-GAL, but differences probably exist between individual and group maturation.

Physiology of digestion and the molecular characterization of the major digestive enzymes from Periplaneta americana.

Cockroaches are among the first insects to appear in the fossil record. This work is part of ongoing research on insects at critical points in the evolutionary tree to disclose evolutionary trends in the digestive characteristics of insects. A transcriptome (454 Roche platform) of the midgut of Periplanetaamericana was searched for sequences of digestive enzymes. The selected sequences were manually curated. The complete or nearly complete sequences showing all characteristic motifs and highly expressed (reads counting) had their predicted sequences checked by cloning and Sanger sequencing. There are two chitinases (lacking mucin and chitin-binding domains), one amylase, two α- and three ß-glucosidases, one ß-galactosidase, two aminopeptidases (none of the N-group), one chymotrypsin, 5 trypsins, and none ß-glucanase. Electrophoretic and enzymological data agreed with transcriptome data in showing that there is a single ß-galactosidase, two α-glucosidases, one preferring as substrate maltase and the other aryl α-glucoside, and two ß-glucosidases. Chromatographic and enzymological data identified 4 trypsins, one chymotrypsin (also found in the transcriptome), and one non-identified proteinase. The major digestive trypsin is identifiable to a major P. americana allergen (Per a 10). The lack of ß-glucanase expression in midguts was confirmed, thus lending support to claims that those enzymes are salivary. A salivary amylase was molecularly cloned and shown to be different from the one from the midgut. Enzyme distribution showed that most digestion occurs under the action of salivary and midgut enzymes in the foregut and anterior midgut, except the posterior terminal digestion of proteins. A counter-flux of fluid may be functional in the midgut of the cockroach to explain the low excretory rate of digestive enzymes. Ultrastructural and immunocytochemical localization data showed that amylase and trypsin are released by both merocrine and apocrine secretion mainly from gastric caeca. Finally, a discussion on Polyneoptera digestive physiology is provided.

miR128 up-regulation correlates with impaired amyloid ß(1-42) degradation in monocytes from patients with sporadic Alzheimer's disease.

Alzheimer's disease (AD), the most common form of dementia in elderly individuals, is characterized by neurofibrillary tangles, extracellular amyloid-ß (Aß) plaques and neuroinflammation. New evidence has shown that the lysosomal system might be a crossroad in which etiological factors in AD pathogenesis converge. This study shows that several lysosomal enzymes, including Cathepsin B, D, S, ß-Galactosidase, α-Mannosidase, and ß-Hexosaminidase, were less expressed in monocytes and lymphocytes from patients with a clinical diagnosis of AD dementia compared with cells from healthy controls. In vitro experiments of gain and loss of function suggest that down-regulation is a direct consequence of miR-128 up-regulation found in AD-related cells. The present study also demonstrates that miR-128 inhibition in monocytes from AD patients improves Aß(1-42) degradation. These results could contribute to clarify the molecular mechanisms that affect the imbalanced Aß production/clearance involved in the pathogenesis of AD.

Inhibition of oxidative stress in rostral ventrolateral medulla improves impaired baroreflex sensitivity in stroke-prone spontaneously hypertensive rats.

Reactive oxygen species (ROS) in rostral ventrolateral medulla (RVLM) of brainstem contribute to sympathoexcitation and are critically involved in the pathogenesis of hypertension. Baroreflex sensitivity (BRS) is a valuable prognostic parameter of the autonomic nervous system, and is impaired in hypertension. The aim of the present study was to determine whether or not a chronic reduction of ROS in the RVLM improves impaired BRS in hypertensive rats. We transfected adenovirus vectors encoding either manganese superoxide dismutase (AdMnSOD) or ß-galactosidase (AdLacZ) into the RVLM of stroke-prone spontaneously hypertensive rats (SHRSP). We measured BRS using the spontaneous sequence method. BRS was significantly lower in SHRSPs than in Wistar-Kyoto rats. In the AdMnSOD-transfected SHRSP, blood pressure, heart rate, and sympathetic nervous system activation were significantly decreased from day 5 after the gene transfer. BRS in the AdMnSOD-transfected SHRSP was significantly increased from day 4 after the gene transfer with the reduction of ROS in the RVLM. Furthermore, in the AdMnSOD-transfected SHRSP, intravenous infusion of atropine dramatically decreased BRS. In contrast, in the AdLacZ-transfected SHRSP, atropine did not decrease BRS. These results suggest that chronic reduction of ROS in the local RVLM improves the impaired BRS in SHRSP through inhibition of the sympathetic component.

Regenerative treatment using a radioelectric asymmetric conveyor as a novel tool in antiaging medicine: an in vitro beta-galactosidase study.

BACKGROUND: Beta-galactosidase is the most widely used biomarker for highlighting the processes of cellular aging, including neurodegeneration. On this basis, we decided to test in vitro whether a set of rescuing/reparative events previously observed by us in subjects treated with radioelectric asymmetric conveyor (REAC) technology may also involve antagonism of a marker of aging-related degenerative processes, as assessed by a reduction in beta-galactosidase at the cellular level. METHODS: Human adipose-derived stem cells were cultured at different passages, ranging from 5 to 20, with or without REAC exposure for 12 hours. The cells were then processed for biochemical beta-galactosidase staining and morphological microscopy analysis. RESULTS: We observed a significant reduction in expression of senescence associated-beta-galactosidase, and a persistence of fibroblast-like morphology typical of human adipose-derived stem cells, even at late passages. CONCLUSION: Our results indicate the ability of REAC technology to counteract in vitro senescence of human adipose-derived stem cells, and prompt the hypothesis that such technology may be exploited to antagonize in vivo senescence of tissue-resident or transplanted stem cells playing an important role in clinical treatment of age-related processes.

AFos inhibits phenylephrine-mediated contractile dysfunction by altering phospholamban phosphorylation.

Using neonatal rat ventricular myocytes, we previously reported that the expression of a dominant negative form of the c-Fos proto-oncogene (AFos) inhibited activator protein 1 activity and blocked the induction of the pathological gene profile stimulated by phenylephrine (PE) while leaving growth unaffected. We now extend these observations to the adult rat ventricular myocyte (ARVM) to understand the relationship between gene expression, growth, and function. Ventricular myocytes were isolated from adult rats and infected with adenovirus expressing beta-galactosidase (control) or AFos. The cells were subsequently treated with PE, and protein synthesis, gene program, calcium transients, and contractility were evaluated. As seen with the neonatal rat ventricular myocytes, in control cells PE stimulated an increase in protein synthesis, induced the pathological gene profile, and exhibited both depressed contractility and calcium transients. Although ARVMs expressing AFos still had PE-induced growth, pathological gene expression as well as contractility and calcium handling abnormalities were inhibited. To determine a possible mechanism of the preserved myocyte function in AFos-expressing cells, we examined phospholamban (PLB) and sarco(endo)plasmic reticulum calcium-ATPase proteins. Although there was no change in total PLB or sarco(endo)plasmic reticulum calcium-ATPase expression in response to PE treatment, PE decreased the phosphorylation of PLB at serine-16, an observation that was prevented in AFos-expressing cells. In conclusion, although PE-induced growth was unaffected in AFos-expressing ARVMs, the expression of the pathological gene profile was inhibited and both contractile function and calcium cycling were preserved. The inhibition of functional deterioration was, in part, due to the preservation of PLB phosphorylation.

The novel epididymis-specific beta-galactosidase-like gene Glb1l4 is essential in epididymal development and sperm maturation in rats.

We describe a novel epididymis-specific cDNA named Glb1l4, which was isolated from rat epididymis by differential display of mRNAs. Glb1l4 cDNA contains 2607 nucleotides and encodes a 637-amino acid protein with 50% similarity to mouse beta-galactosidase. The gene is located on chromosome 8q13, spanning 21 exons. Northern blot analysis reveals that Glb1l4 is specifically expressed in the caput region of epididymis and upregulated by androgen. A specific polyclonal antiserum against the N-terminal peptide of GLB1L4 has been produced. Western blot analysis and immunohistochemistry assay reveal that GLB1L4 is specifically expressed in the principal cells of the caput epididymis. Interestingly, its expression peaks at Postnatal Day 45 in mRNA level and at Postnatal Day 60 in protein level while the epididymis column cells undergo differentiation. Moreover, within this very period this secretory protein is confined inside the cell with a change of subcellular distribution pattern, which implies its important roles in the cell differentiation process. Only after the epididymal epithelium differentiation is completed and the spermatozoa enter the epididymal lumen is the GLB1L4 secreted into the luminal fluid and bound on the sperm head. Our results suggest that GLB1L4 may play various roles in principal cell differentiation and sperm maturation.

Endogenous beta-glucosidase and beta-galactosidase activities from selected probiotic micro-organisms and their role in isoflavone biotransformation in soymilk.

AIM: To compare endogenous beta-glucosidases and beta-galactosidases for hydrolysis of the predominant isoflavone glycosides into isoflavone aglycones in order to improve biological activity of soymilk. METHODS AND RESULTS: beta-glucosidase and beta-galactosidase activities of probiotic organisms including Lactobacillus acidophilus ATCC 4461, Lactobacillus casei 2607 and Bifidobacterium animalis ssp. lactis Bb12 in soymilk were evaluated and correlated with the increase in concentration of isoflavone aglycones during fermentation. The concentrations of isoflavone compounds in soymilk were monitored using a Varian model high-performance liquid chromatography (HPLC) with an amperometric electrochemical detector. In all micro-organisms, beta-glucosidase activity was found greater than that of beta-galactosidase. There was an increase in the aglycone concentration with incubation time because of the apparent hydrolytic action on isoflavone glycosides. Aglycone concentration in the soymilk with L. acidophilus 4461, L. casei 2607 and B. animalis ssp. lactis Bb12, increased by 5.37-, 5.52- and 6.10-fold, respectively, after 15 h of fermentation at 37 degrees C. The maximum hydrolytic potential was also observed at 15 h of fermentation for the three micro-organims coinciding with peak activities of the two enzymes. CONCLUSIONS: beta-glucosidase activity was more than 15 times higher than beta-galactosidase activity in soymilk for each of the micro-organisms during fermentation. beta-glucosidase played a greater role in isoflavone glycoside hydrolysis. SIGNIFICANCE AND IMPACT OF THE STUDY: Screening for beta-glucosidase and beta-galactosidase activities among probiotics in soymilk is important for the improvement of biological activity of soymilk and in the selection of micro-organisms for use in the growing industry of functional foods and beverages.

Senescence as an anticancer mechanism.

Senescence was originally described as a terminal nondividing state of normal human cells reached after many cell divisions in culture. The cause was shown to be shortening of telomeres, leading to telomere dysfunction and cell cycle arrest. Subsequently, a more rapid, nontelomere-dependent form of senescence, often termed stress-induced premature senescence, was described. Mostly importantly, it occurs in response to activated oncogene products. Oncogene-induced senescence has been shown to play a role in tumor suppression in vivo; it does not seem to involve changes in telomeres. A second phenomenon that plays a role in tumor suppression, which does involve progressive telomere shortening, is crisis, the state that cells reach when cell cycle checkpoints are impaired and cells can no longer respond to telomere shortening or oncogene activation by entering senescence. These two processes, oncogene-induced senescence and telomere-based crisis, exert powerful anticancer effects.

Functional genomic analysis of Arabidopsis thaliana glycoside hydrolase family 35.

Catalysing the hydrolysis of terminal beta-galactosyl residues from carbohydrates, galactolipids, and glycoproteins, glycoside hydrolase family 35 (beta-galactosidases; BGALs) are widely distributed in plants and believed to play many key roles, including modification of cell wall components. Completion of the Arabidopsis thaliana genome sequencing project has, for the first time, allowed an examination of the total number, gene structure, and evolutionary patterns of all Family 35 members in a representative (model) angiosperm. Reiterative database searches established a multigene family of 17 members (designated BGAL1-BGAL17). Using these genes as query sequences, BLAST and Hidden Markov Model searches identified BGAL genes among 22 other eukaryotes, whose genomic sequences are known. The Arabidopsis (n=17) and rice (n=15) BGAL families were much larger than those of Chlamydomonas, fungi, and animals (n=0-4), and a lineage-specific expansion of BGAL genes apparently occurred after divergence of the Arabidopsis and rice lineages. All plant BGAL genes, with the exception of Arabidopsis BGAL17 and rice Os 9633.m04334, form a monophyletic group. Arabidopsis BGAL expression levels are much higher in mature leaves, roots, flowers, and siliques but are lower in young seedlings. BGAL8, BGAL11, BGAL13, BGAL14, and BGAL16 are expressed only in flowers. Catalytically active BGAL4 was produced in the E. coli and baculoviral expression systems, purified to electrophoretic homogeneity, and partially characterized. The purified enzyme hydrolyzed p- and o-nitrophenyl-beta-d-galactosides. It also cleaved beta-(1,3)-, beta-(1,4)-, and beta-(1,6)-linked galactobiosides and galactotriosides, showing a marked preference for beta-(1,3)- and beta-(1,4)-linkages.

Overexpression and functional analysis of cold-active beta-galactosidase from Arthrobacter psychrolactophilus strain F2.

Cold-active beta-galactosidase from Arthrobacter psychrolactophilus strain F2 was overexpressed in Escherichia coli using the Cold expression system and the recombinant enzyme, rBglAp, was characterized. The purified rBglAp exhibited similar enzymatic properties to the native enzyme, e.g., (i) it had high activity at 0 degrees C, (ii) its optimum temperature and pH were 10 degrees C and 8.0, respectively, and (iii) it was possible to rapidly inactivate the rBglAp at 50 degrees C in 5 min. Moreover, rBglAp was able to hydrolyze both ONPG and lactose with K(m) values of 2.7 and 42.1mM, respectively, at 10 degrees C. One U of rBglAp could hydrolyze about 70% of the lactose in 1 ml of milk in 24h, and the enzyme produced trisaccharide from lactose. We conclude that rBglAp is a cold-active enzyme that is extremely heat labile and has significant potential application to the food industry.

Binding of elastin peptides to S-Gal protects the heart against ischemia/reperfusion injury by triggering the RISK pathway.

Elastin peptides (EPs) generated by hydrolysis of elastic fibers by elastinolytic enzymes display a wide spectrum of biological activities. Here, we investigated their influence on rat heart ischemia-mediated injury using the Langendorff ex vivo model. EPs, i.e., kappa elastin, at 1.32- and 660-nM concentrations, when administered before the ischemia period, elicited a beneficial influence against ischemia by accelerating the recovery rate of heart contractile parameters and by decreasing significantly creatine kinase release and heart necrosis area when measured at the onset of the reperfusion. All effects were S-Gal-dependent, as being reproduced by (VGVAPG)3 and as being inhibited by receptor antagonists, such as lactose and V14 peptide (VVGSPSAQDEASPL). EPs interaction with S-Gal triggered NO release and activation of PI3-kinase/Akt and ERK1/2 in human coronary endothelial cells (HCAECs) and rat neonatal cardiomyocytes (RCs). This signaling pathway, as designated as RISK, for reperfusion injury salvage kinase pathway, was shown to be responsible for the beneficial influence of EPs on ischemia/reperfusion injury on the basis of its inhibition by specific pharmacological inhibitors. EPs survival activity was attained at a concentration averaging that present into the blood circulation, supporting the contention that these matrikines might offer a natural protection against cardiac injury in young and adult individuals. Such protective effect might be lost with aging, since we found that hearts from 24-month-old rats did not respond to EPs.

Ectopically hTERT expressing adult human mesenchymal stem cells are less radiosensitive than their telomerase negative counterpart.

During the past several years increasing evidence indicating that the proliferation capacity of mammalian cells is highly radiosensitive, regardless of the species and the tissue of origin of the cells, has accumulated. It has also been shown that normal bone marrow cells of mice have a similar radiosensitivity to other mammalian cells so far tested. In this study, we investigated the genetic effects of ionizing radiation (2.5-15 Gy) on normal human mesenchymal stem cells and their telomerised counterpart hMSC-telo1. We evaluated overall genomic integrity, DNA damage/repair by applying a fluorescence-detected alkaline DNA unwinding assay together with Western blot analyses for phosphorylated H2AX and Q-FISH was applied for investigation of telomeric damage. Our results indicate that hMSC and TERT-immortalized hMSCs can cope with relatively high doses of gamma-rays and that overall DNA repair is similar in the two cell lines. The telomeres were extensively destroyed after irradiation in both cell types suggesting that telomere caps are especially sensitive to radiation. The TERT-immortalized hMSCs showed higher stability at telomeric regions than primary hMSCs indicating that cells with long telomeres and high telomerase activity have the advantage of re-establishing the telomeric caps.