Spontaneous reversal of the developmental aging of normal human cells following transcriptional reprogramming.

AIM: To determine whether transcriptional reprogramming is capable of reversing the developmental aging of normal human somatic cells to an embryonic state. MATERIALS & METHODS: An isogenic system was utilized to facilitate an accurate assessment of the reprogramming of telomere restriction fragment (TRF) length of aged differentiated cells to that of the human embryonic stem (hES) cell line from which they were originally derived. An hES-derived mortal clonal cell strain EN13 was reprogrammed by SOX2, OCT4 and KLF4. The six resulting induced pluripotent stem (iPS) cell lines were surveyed for telomere length, telomerase activity and telomere-related gene expression. In addition, we measured all these parameters in widely-used hES and iPS cell lines and compared the results to those obtained in the six new isogenic iPS cell lines. RESULTS: We observed variable but relatively long TRF lengths in three widely studied hES cell lines (16.09-21.1 kb) but markedly shorter TRF lengths (6.4-12.6 kb) in five similarly widely studied iPS cell lines. Transcriptome analysis comparing these hES and iPS cell lines showed modest variation in a small subset of genes implicated in telomere length regulation. However, iPS cell lines consistently showed reduced levels of telomerase activity compared with hES cell lines. In order to verify these results in an isogenic background, we generated six iPS cell clones from the hES-derived cell line EN13. These iPS cell clones showed initial telomere lengths comparable to the parental EN13 cells, had telomerase activity, expressed embryonic stem cell markers and had a telomere-related transcriptome similar to hES cells. Subsequent culture of five out of six lines generally showed telomere shortening to lengths similar to that observed in the widely distributed iPS lines. However, the clone EH3, with relatively high levels of telomerase activity, progressively increased TRF length over 60 days of serial culture back to that of the parental hES cell line. CONCLUSION: Prematurely aged (shortened) telomeres appears to be a common feature of iPS cells created by current pluripotency protocols. However, the spontaneous appearance of lines that express sufficient telomerase activity to extend telomere length may allow the reversal of developmental aging in human cells for use in regenerative medicine.

Insulin-like growth factor-1 receptor immunoreactive cells are selectively maintained in the paraventricular hypothalamus of calorically restricted mice.

The mammalian lifespan is dramatically extended by both caloric restriction (CR) and insulin-like growth factor-1 (IGF-1) suppression. Both interventions involve neuroendocrine alterations directed by the hypothalamus. Yet, it remains unclear whether CR exerts its affects by altering central IGF-1 sensitivity. With this question in mind, we investigated the influence of CR and normal aging on hypothalamic IGF-1 sensitivity, by measuring the changes in IGF-1 receptor (IGF-1R) populations. Taking IGF-1 receptor (IGF-1R) immunoreactivity as an index of sensitivity to IGF-1, we counted IGF-1R immunoreactive and non-immunoreactive cells in the paraventricular nucleus (PVN) of Young-ad libitum fed (Young-Al, 6 weeks old), Old-ad libitum fed (Old-Al, 22 months old), and old calorically restricted (Old-CR, 22 months old) female B6D2F1 mice. An automated imaging microscopy system (AIMS) was used to generate cell counts for each cross-section of PVN hypothalamus. Ad libitum fed mice show a 37% reduction in IGF-1R immunoreactive cells and a 12% reduction in the total cell population of the PVN with aging. In comparison, caloric-restricted mice show a 33% reduction in IGF-1R immunoreactive cells and a notable 24% decrease in the total cell population with aging. This selective maintenance of IGF-1R expressing cells coupled with the simultaneous loss of non-immunoreactive cells, results in a higher percentage of IGF-1R immunoreactive cells in the PVNs of CR mice. Thus, the decline in the percentage of IGF-1 sensitive cells in the PVN with age is attenuated by CR.

Age-dependent loss of insulin-like growth factor-1 receptor immunoreactive cells in the supraoptic hypothalamus is reduced in calorically restricted mice.

Both life-long caloric restriction (CR) and the suppression of insulin-like growth factor-1 (IGF-1) signaling reliably extend the mammalian lifespan. The neuroendocrine system, regulated by the hypothalamus, remains the most convincing site of action for both these modes of life extension. Yet, determining whether CR actions are mediated by the modulation of neuroendocrine IGF-1 signaling remains unclear. Of the hypothalamic nuclei that express the IGF-1 receptor (IGF-1R), the cells of the supraoptic nucleus (SON) display some of the most robust IGF-1R expression. Taking IGF-1R immunoreactivity as an index of sensitivity to IGF-1, we counted IGF-1R immunoreactive and non-immunoreactive cells in the SON of young-ad-libitum fed (young-Al, 6 weeks), old-ad-libitum fed (Old-Al, 22 months), and old-calorie-restricted (Old-CR, 22 months) female B6D2F1 mice. An automated imaging microscopy system (AIMS) was used to generate cell counts for each section of supraoptic hypothalamus. Results show that while the total number of cells in the SON of ad-libitum fed mice does not change significantly with aging, a significant reduction in IGF-1R immunoreactive cells does occur in ad-libitum fed mice with aging. In contrast to this, calorie restricted mice show both a decline in the total number of cells and IGF-1R immunoreactive cells in the SON with age, but with the decrease in the latter being notably attenuated when compared to the degree of loss seen in ad-libitum fed mice. Thus, while CR induces greater loss in the total number of cells in the SON with age, it reduces the degree of age-dependent loss seen in IGF-1R expressing cells. As a result, when compared to Old-AL mice, the SON of Old-CR mice displays a greater proportion of IGF-1R cells and thus possibly enhanced IGF-1 sensitivity with aging.

Efficient prenylation by a plant geranylgeranyltransferase-I requires a functional CaaL box motif and a proximal polybasic domain.

Geranylgeranyltransferase-I (GGT-I) is a heterodimeric enzyme that shares a common alpha-subunit with farnesyltransferase (FTase) and has a distinct beta-subunit. GGT-I preferentially modifies proteins, which terminate in a CaaL box sequence motif. Cloning of Arabidopsis GGT-I beta-subunit (AtGGT-IB) was achieved by a yeast (Saccharomyces cerevisiae) two-hybrid screen, using the tomato (Lycopersicon esculentum) FTase alpha-subunit (FTA) as bait. Sequence and structure analysis revealed that the core active site of GGT-I and FTase are very similar. AtGGT-IA/FTA and AtGGT-IB were co-expressed in baculovirus-infected insect cells to obtain recombinant protein that was used for biochemical and molecular analysis. The recombinant AtGGT-I prenylated efficiently CaaL box fusion proteins in which the a(2) position was occupied by an aliphatic residue, whereas charged or polar residues at the same position greatly reduced the efficiency of prenylation. A polybasic domain proximal to the CaaL box motif induced a 5-fold increase in the maximal reaction rate, and increased the affinity of the enzyme to the protein substrate by an order of magnitude. GGT-I retained high activity in a temperature range between 24 degrees C and 42 degrees C, and showed increased activity rate at relatively basic pH values of 7.9 and 8.5. Reverse transcriptase-polymerase chain reaction, protein immuno-blots, and transient expression assays of green fluorescent protein fusion proteins show that GGT-IB is ubiquitously expressed in a number of tissues, and that expression levels and protein activity were not changed in mutant plants lacking FTase beta-subunit.

Altered circadian rhythm of blood pressure in shift workers.

The effect of shift work on the circadian rhythm of blood pressure (BP) was studied in male bakery workers. The study group consisted of 28 men, blue collar non-rotating shift workers, 20-60 years of age, and the control group comprised 30 men, blue collar, day workers in the same age group. BP was evaluated in all subjects by 24h BP monitoring. Day workers showed typical circadian rhythm with a drop in both systolic and diastolic BP at night. This pattern was reversed in night workers. The peak SBP for night workers was at 11 pm and among day workers at 4 pm. Peak DBP was recorded among night workers at 10 pm and among day workers at 3 pm. All subjects showed a highly significant cyclic variation in BP. Whereas the range for SBP was similar in these two age groups (P > 0.05), the amplitude of DBP tended to be smaller in young workers. Therapeutic decisions for night shift workers with hypertension should take into account their altered BP cycle.

Low temperature preservation and space medicine.

Cold maintenance may be an option for compromised space-borne astronauts. Contemporary aneurysm surgery can involve cooling below 20 degrees C for nearly one hour. Dogs and baboons have survived blood-substituted hypothermia for 1-3 hours. Hamsters have recovered from partial-freezing below -1 degree C, and supercooling at -5 degrees C. Laboratory frogs have survived partial-freezing from -9 degrees C, while in nature, frogs may overwinter in these states. While some invertebrates can tolerate freezing to cryogenic temperatures, no vertebrate has survived complete freezing. The following studies (hypothermia and sub-zero experiments) were conducted to explore low temperature preservation of rodents, dogs and baboons.

Variability and uniformity of mitochondrial DNA in populations of putative diploid ancestors of common wheat.

By using restriction endonuclease digestion patterns, the degree of intraspecific polymorphism of mitochondrial DNA in four diploid species of wheat and Aegilops, Ae. speltoides, Ae. longissima, Ae. squarrosa, and Triticum monococcum, was assessed. The outbreeding Ae. speltoides was found to possess the highest degree of variability, the mean number of nucleotide substitutions among conspecific individuals being 0.027 substitutions per nucleotide site. A very low degree of mtDNA variation was detected among Ae. longissima accessions, with most of the enzyme-probe combinations exhibiting uniform hybridization patterns. The mean number of substitutions among Ae. longissima individuals was 0.001 substitutions per nucleotide site. The domesticated diploid wheat T. monococcum var. monococcum and its conspecific variant T. monococcum var. boeoticum seem to lack mitochondrial DNA variability altogether. Thus, the restriction fragment pattern can be used as a characteristic identifier of the T. monococcum cytoplasmic genome. Similarly, Ae. squarrosa accessions were found to be genetically uniform. A higher degree of variation among accessions is observed when noncoding sequences are used as probes then when adjacent coding regions are used. Thus, while noncoding regions may contain regulatory functions, they are subject to less stringent functional constraints than protein-coding regions. Intraspecific variation in mitochondrial DNA correlates perfectly with the nuclear variability detected by using protein electrophoretic characters. This correlation indicates that both types of variation are selectively neutral and are affected only by the effective population size.

Family of human neuronal external surface epitopes defined by Torpedo monoclonal antibodies.

We are employing a library of monoclonal antibodies (MAbs) that were made to Torpedo cholinergic synaptosomes to identify conserved, physiologically vital epitopes of the neuronal surface. Our particular interest is in those epitopes that are present on some but not all neurons. In the present study we screened this library on different cell lines, the neuronal cell lines PC12, NG108, MC-IXC, and SY5Y, and the endocrine cell lines GH-3 and HIT. Of these cell lines, only SY5Y cells bind MAbs that define neuronal surface subsets. Utilizing its parent cell line, SK-N-SH, we verified that six MAbs, Tor 25, Tor 103, Tor 190, Tor 201, Tor 219, and Tor 233, bind the external neuronal surface. The cytolocalization of all six MAbs is very similar: the membrane of the cell body and its processes are finely outlined in a punctate distribution. Western blot analyses of Torpedo electric organ homogenates, a highly enriched source of antigenic material, revealed that each MAb identifies multiple polypeptides, two of which have the relative mobilities of 180 kD and 67 kD. In a screen of peripheral nerves from cases of amyotrophic lateral sclerosis (ALS), we found that all these MAbs revealed surface alterations; some displayed a decrease in binding, while others displayed an increase. The combined data provide evidence that these epitopes belong to an important, complex family of polypeptides of the external neuronal surface.

Doxorubicin affects tau protein metabolism in human neuroblastoma cells.

The microtubule associated protein called tau, found primarily in neurons, was detected in a human neuroblastoma cell line, LAN-5. Cells treated with retinoic acid (2.0 x 10(-5) M) differentiate and acquire processes similar to neurons. Differentiated and logarithmically growing undifferentiated cells were exposed to varying doses of doxorubicin (an anthracycline chemotherapeutic antibiotic). While doxorubicin was lethal to many undifferentiated dividing cells, it was not as damaging to differentiated cells. After 2 to 4 days of doxorubicin treatment, the cells were harvested, the protein concentration determined and SDS-PAGE performed. Proteins were blotted onto nitrocellulose paper and immunostained with either a rabbit antiserum or mouse monoclonal antibody to tau. Undifferentiated LAN-5 cells treated with 4.0 x 10(-8) M doxorubicin for 4 days and cells treated with 8.0 x 10(-8) M doxorubicin for 2 days displayed a distinct lower band (just below the 50 kd marker) that was either absent or very faint in untreated controls.

Similarities and differences between tau protein and chromobindin A.

Tau protein and Chromobindin A have several features in common but are not identical. Both consist of a small group of closely related proteins which can form aggregates. Both have a similar range of molecular weights (53-62 kDa) and isoelectric points (6.0-7.5). While Chromobindin A is known to be membrane associated, there is evidence that Tau protein also interacts with phospholipids. Both, not present in all tissues, can be found in the adrenal medulla. Despite these similarities both classes of proteins are unique and immunologically distinct. A rabbit antisera to Tau does not cross react with Chromobindin A. In addition, while protein kinase C and Ca/Calmodulin-dependent protein kinase II phosphorylate Tau protein, they do not phosphorylate Chromobindin A, demonstrating the specificity of these kinases for Tau protein phosphorylation.

A human teratocarcinoma which expresses a rare neuronal cell surface antigen.

A molecular characterization of the events at the cell surface of neurons is pivotal for our understanding of how the nervous system is formed and maintained. This study of cell surface events in the human nervous system may be crucial to the study of human neurological maladies. NTERA-2 is a human teratocarcinoma which is unique amongst the teratocarcinomas for its ability to express many neurons. Tested for the binding of a monoclonal antibody Tor 23, which recognizes a surface antigen on rare and specific neurons, cultures of NTERA-2 cells contained cells with a neuronal morphology which bound the monoclonal antibody Tor 23 on their surface. The data indicate that Tor 23 antigen is a cell surface molecule with the same or very similar properties in rays, rats, and humans. The NTERA-2 cells are thus capable of expressing highly differentiated neuronal cell surface phenotypes and promise to be a powerful model system for the study of cell surface events of the human nervous system in vitro.

Glutamic acid decarboxylase activity in discrete hypothalamic nuclei during the development of rats.

We have assessed the activity (nmol/mg protein/h) of glutamic acid decarboxylase (GAD) in discrete hypothalamic nuclei before and after sexual maturation in the developing female rat. Activity in other brain regions including the cortex, septum and caudate-putamen was also assessed. While there appears to be a general rise (approximately 30%), with age, in GAD activity, the rise is most marked, and highly significant (P less than 0.001), in the anterior portion of the hypothalamus (56%). In contrast, no significant increase of GAD activity was found in the medical basal hypothalamus.

Monoclonal antibody Tor 23 recognizes a determinant of a presynaptic acetylcholinesterase.

A significant proportion of the acetylcholinesterase that is present in the electric organ of Torpedo californica exists as a presynaptic membrane molecule. The monoclonal antibody Tor 23 binds the Torpedo presynaptic nerve membrane where it recognizes a polypeptide of 68,000 daltons. Our present studies indicate that Tor 23 identifies acetylcholinesterase. From the homogenates of Torpedo nerve terminals, Tor 23 immunoprecipitates measurable esterase activity. Esterase precipitation was not observed with no Tor 23 added; nor was it observed with any other test antibodies, including other Tor antibodies, in particular, Tor 70, which binds, as does Tor 23, to the presynaptic nerve membrane. The esterase activity was specific for acetylcholinesterase. Our studies indicate the molecule defined by Tor 23 has the solubility properties described for that of presynaptic acetylcholinesterase: it is soluble in detergent-treated electroplax homogenates and insoluble in high-salt extractions. In sections of Torpedo back muscle, both nerve and endplate acetylcholinesterase can be detected histochemically. Tor 23 localizes to the nerve and is not clustered at the endplate. The utility of the antibody Tor 23 thus includes biochemical and histological analyses of the multiple forms of acetylcholinesterase.

Effects of dietary choline on memory and brain chemistry in aged mice.

The purpose of this study was to investigate in more detail the characteristics of the age-related extension of the retrograde amnesia gradient previously demonstrated in a passive avoidance task [6]. In Experiment 1, it was found that while 2-3 month old mice were susceptible to the amnesic effects of anisomycin (ANI) only when given prior to 15 min post-training, memory of 14-16 month old mice was susceptible to disruption when ANI was given as late as 20 min post-training, and retention of 17-20 month old mice was impaired when ANI was injected even as late as 30 min after training. Experiment 2 examined whether the age-related change in susceptibility to the effects of ANI could be ameliorated by chronic pretreatment with a choline-enriched diet. Results showed that ANI injected 20 min after training did not induce amnesia in choline treated mice (14.5 month old), but did induce amnesia when injected 15 min post training. Subsequent assay of choline acetyltransferase (ChAT) and tyrosine hydroxylase (TH) activity showed that choline treatment significantly reduced ChAT activity but did not affect TH activity. It appears that dietary choline treatment can render new long-term memories less susceptible to disruption following training.

The influence of neurite-inducing agents on cholinergic activity in human neuroblastoma cells.

Various factors capable of inducing morphological differentiation of neuroblastoma cells were studied to determine whether they may also specifically regulate cholinergic function in human cholinergic neuroblastoma cells MC-IXC. Choline acetyltransferase (CAT) activity was monitored to reflect cholinergic function while Mg(2+) ATPase activity was monitored to reflect effects which are not related specifically to neuronal function. Among the agents, dimethylsulfoxide (DMSO), dimethylformamide (DMF), dimethylacetamide (DMA), D-α-tocopherol, ascorbic acid and dibutyryl-cAMP, only low doses of DMSO were found to selectively inhibit CAT activity while Mg(2+) ATPase activity along with population growth remained unaffected. Of the three chemically related organic compounds tested (DMSO, DMF, DMA), DMSO was the least potent with regard to inhibition of population growth and Mg(2+) ATPase activity while DMA was the most potent inhibitor. Sodium butyrate caused a decline in CAT activity while retinoic acid induced an enhancement. The combination of sodium butyrate with retinoic acid caused an enhancement of CAT activity similar in magnitude to that observed with retinoic acid alone. Lastly, CAT activity was found to be independent of population density unlike that in different neuroblastoma cell lines.(8,12,19.)

The dependence of the molecular dynamics of calmodulin upon pH and ionic strength.

The mobilities of several fluorescent probes placed at different locations on calmodulin in the absence of Ca2+ have been found to depend upon the charge, ionic strength, and temperature. In general, the time decay of fluorescence anisotropy could be fitted with two rotational correlation times. The shorter of these reflects primarily the motion of the probe itself, while the longer corresponds to the motion of a major portion of the molecule. An increase in ionic strength or a decrease in net charge results in a decrease in the relative amplitude of the shorter correlation time, while an increase in temperature produces an increase in its amplitude. These results are consistent with, and suggest, that an increase in probe mobility accompanies an expansion of the calmodulin molecule under conditions of high electrostatic stress.

Aging and sister chromatid exchange. VII. Effect of aging on background SCE in vivo.

The frequency of background or spontaneous sister chromatid exchanges (SCE) was estimated in young and old rat and mouse bone marrow cell populations by exposing these cells to increasing concentrations of bromodeoxyuridine (BrdU). At the lowest levels of BrdU where SCE could be accurately assessed, there was no significant difference in background SCE between young and old cell populations. Extrapolation to zero BrdU concentration yielded SCE frequencies of approximately 1.0 SCE/cell/cell cycle in both mouse and rat cells.