How cells dedifferentiate: a lesson from plants

The remarkable regenerative capacity displayed by plants and various vertebrates, such as amphibians, is largely based on the capability of somatic cells to undergo dedifferentiation. In this process, mature cells reverse their state of differentiation and acquire pluripotentiality--a process preceding not only reentry into the cell cycle but also a commitment for cell death or trans- or redifferentiation. Recent studies provide a new perspective on cellular dedifferentiation, establishing chromatin reorganization as its fundamental theme.     

Characterization of Arabidopsis thaliana methyl-CpG-binding domain (MBD) proteins

Cytosine methylation at symmetrical CpG and CpNpG sequences plays a key role in the epigenetic control of plant growth and development; yet, the way by which the methylation signal is interpreted into a functional state has not been elucidated. In animals, the methylation signal is recognized by methyl-CpG-binding domain (MBD) proteins that specifically bind methylated CpG dinucleotides. In Arabidopsis thaliana, 12 putative MBD proteins were identified and classified into seven subclasses. Here, we characterized six MBD proteins representing four subclasses (II, III, IV, and VI) of the Arabidopsis MBD family. We found that AtMBD7 (subclass VI), a unique protein containing a double MBD motif, as well as AtMBD5 and AtMBD6 (subclass IV), bind specifically symmetrically methylated CpG sites. The MBD motif derived from AtMBD6, but not from AtMBD2, was sufficient for binding methylated CpG dinucleotides. AtMBD6 precipitated histone deacetylase (HDAC) activity from the leaf nuclear extract. The examined AtMBD proteins neither bound methylated CpNpG sequences nor did they display DNA demethylase activity. Our results suggest that AtMBD5, AtMBD6, and AtMBD7 are likely to function in Arabidopsis plants as mediators of the CpG methylation, linking DNA methylation-induced gene silencing with histone deacetylation.     

Variability in Tuberculosis Granuloma T Cell Responses Exists,but a Balance of Pro- and Anti-inflammatory Cytokines Is Associated with Sterilization

Lung granulomas are the pathologic hallmark of tuberculosis (TB). T cells are a major cellular component of TB lung granulomas and are known to play an important role in containment of Mycobacterium tuberculosis (Mtb) infection. We used cynomolgus macaques, a non-human primate model that recapitulates human TB with clinically active disease, latent infection or early infection, to understand functional characteristics and dynamics of T cells in individual granulomas. We sought to correlate T cell cytokine response and bacterial burden of each granuloma, as well as granuloma and systemic responses in individual animals. Our results support that each granuloma within an individual host is independent with respect to total cell numbers, proportion of T cells, pattern of cytokine response, and bacterial burden. The spectrum of these components overlaps greatly amongst animals with different clinical status, indicating that a diversity of granulomas exists within an individual host. On average only about 8% of T cells from granulomas respond with cytokine production after stimulation with Mtb specific antigens, and few “multi-functional” T cells were observed. However, granulomas were found to be “multi-functional” with respect to the combinations of functional T cells that were identified among lesions from individual animals. Although the responses generally overlapped, sterile granulomas had modestly higher frequencies of T cells making IL-17, TNF and any of T-1 (IFN-γ, IL-2, or TNF) and/or T-17 (IL-17) cytokines than non-sterile granulomas. An inverse correlation was observed between bacterial burden with TNF and T-1/T-17 responses in individual granulomas, and a combinatorial analysis of pair-wise cytokine responses indicated that granulomas with T cells producing both pro- and anti-inflammatory cytokines (e.g. IL-10 and IL-17) were associated with clearance of Mtb. Preliminary evaluation suggests that systemic responses in the blood do not accurately reflect local T cell responses within granulomas.     

Non-peptide alpha(v)beta(3) antagonists. Part 4: potent and orally bioavailable chain-shortened RGD mimetics

Potent non-peptidic alpha(v)beta(3) antagonists have been prepared where deletion of an amide bond from an earlier series of linear RGD-mimetics provides a novel series of chain-shortened alpha(v)beta(3) antagonists with significantly improved oral pharmacokinetics. These chain-shortened alpha(v)beta(3) antagonists represent structurally novel integrin inhibitors.     

The endosomal trafficking regulator LITAF controls the cardiac Nav1.5 channel via the ubiquitin ligase NEDD4-2

The QT interval is a recording of cardiac electrical activity. Previous genome-wide association studies identified genetic variants that modify the QT interval upstream of LITAF (lipopolysaccharide-induced tumor necrosis factor-α factor), a protein encoding a regulator of endosomal trafficking. However, it was not clear how LITAF might impact cardiac excitation. We investigated the effect of LITAF on the voltage-gated sodium channel Nav1.5, which is critical for cardiac depolarization. We show that overexpressed LITAF resulted in a significant increase in the density of Nav1.5-generated voltage-gated sodium current I_Na and Nav1.5 surface protein levels in rabbit cardiomyocytes and in HEK cells stably expressing Nav1.5. Proximity ligation assays showed co-localization of endogenous LITAF and Nav1.5 in cardiomyocytes, whereas co-immunoprecipitations confirmed they are in the same complex when overexpressed in HEK cells. In vitro data suggest that LITAF interacts with the ubiquitin ligase NEDD4-2, a regulator of Nav1.5. LITAF overexpression down-regulated NEDD4-2 in cardiomyocytes and HEK cells. In HEK cells, LITAF increased ubiquitination and proteasomal degradation of co-expressed NEDD4-2 and significantly blunted the negative effect of NEDD4-2 on I_Na. We conclude that LITAF controls cardiac excitability by promoting degradation of NEDD4-2, which is essential for removal of surface Nav1.5. LITAF-knockout zebrafish showed increased variation in and a nonsignificant 15% prolongation of action potential duration. Computer simulations using a rabbit-cardiomyocyte model demonstrated that changes in Ca²⁺ and Na⁺ homeostasis are responsible for the surprisingly modest action potential duration shortening. These computational data thus corroborate findings from several genome-wide association studies that associated LITAF with QT interval variation.     

Effect of shear on morphology,viability and metabolic activity of succinic acid-producing Actinobacillus succinogenes biofilms

Two custom-designed bioreactors were used to evaluate the effect of shear on biofilms of a succinic acid producer, Actinobacillus succinogenes. The first bioreactor allowed for in situ removal of small biofilm samples used for microscopic imaging. The second bioreactor allowed for complete removal of all biofilm and was used to analyse biofilm composition and productivity. The smooth, low porosity biofilms obtained under high shear conditions had an average cell viability of 79% compared to 57% at the lowest shear used. The maximum cell-based succinic acid productivity for high shear biofilm was 2.4 g g⁻¹DCW h⁻¹ compared to the 0.8 g g⁻¹DCW h⁻¹ of the low shear biofilm. Furthermore, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays confirmed higher cell metabolic activities for high shear developed biofilm compared to biofilm developed at low shear conditions. Results clearly indicated that high shear biofilm cultivation has beneficial morphological, viability, and cell-based productivity characteristics.

     

Numerous microRNPs in neuronal cells containing novel microRNAs

Spinal muscular atrophy (SMA) is a common neurodegenerative disease that is caused by deletions or loss-of-function mutations in the Survival of Motor Neuron (SMN) protein. SMN is part of a large complex that functions in the assembly/restructuring of ribonucleoprotein (RNP) complexes. We recently showed in HeLa cells that two components of the SMN complex, Gemin3 and Gemin4, together with the argonaute protein eIF2C2, also associate with microRNAs (miRNAs) as part of a novel class of RNPs termed miRNPs. Here we report on miRNPs isolated from neuronal cell lines of mouse and human, and describe 53 novel miRNAs. Several of these miRNAs are conserved in divergent organisms, including rat, zebrafish, pufferfish, and the nematode Caenorhabditis elegans. The chromosomal locations of most of the novel miRNAs were identified and indicate some phylogenetic conservation of the likely precursor structures. Interestingly the gene locus of one miRNA, miR-175, is a candidate region for two neurologic diseases: early-onset parkinsonism (Waisman syndrome) and X-linked mental retardation (MRX3). Also, several miRNAs identified as part of miRNPs in these cells appear to constitute two distinct subfamilies. These subfamilies comprise multiple copies of miRNAs on different chromosomes, suggesting an important function in the regulation of gene expression.     

Failure-to-Thrive Syndrome Associated with Tumor Formation by Madin–Darby Canine Kidney Cells in Newborn Nude Mice

Tumors that formed in newborn nude mice that were inoculated with 10⁷ Madin–Darby canine kidney (MDCK) cells were associated with a failure-to-thrive (FTT) syndrome consisting of growth retardation, lethargy, weakness, and dehydration. Scoliosis developed in 41% of affected pups. Pups were symptomatic by week 2; severely affected pups became moribund and required euthanasia within 3 to 4 wk. Mice with FTT were classified into categories of mild, moderate, and severe disease by comparing their weight with that of age-matched normal nude mice. The MDCK-induced tumors were adenocarcinomas that invaded adjacent muscle, connective tissue, and bone; 6 of the 26 pups examined had lung metastases. The induction of FTT did not correlate with cell-line aggressiveness as estimated by histopathology or the efficiency of tumor formation (tumor-forming dose 50% endpoint range = 10^2.8 to 10^7.5); however, tumor invasion of the paravertebral muscles likely contributed to the scoliosis noted. In contrast to the effect of MDCK cells, tumor formation observed in newborn mice inoculated with highly tumorigenic, human-tumor–derived cell lines was not associated with FTT development. We suggest that tumor formation and FTT are characteristics of these MDCK cell inocula and that FTT represents a new syndrome that may be similar to the cachexia that develops in humans with cancer or other diseases.Abbreviations: FTT, failure-to-thrive; MDCK, Madin–Darby canine kidney; TPD₅₀, tumor-producing dose log₁₀ 50% endpointThe Madin–Darby canine kidney (MDCK) cell line was established in 1958 from the kidney of a cocker spaniel.^6,16 Since 1962, this cell line has been an important reagent for the isolation and study of influenza viruses^8,22,31 and, more recently, for the development and manufacture of influenza virus vaccines.^3,7,19 MDCK cells are polarized, epithelial cells that exhibit properties of renal tubular epithelium and have been used as in vitro models to evaluate renal tubular functions.^24,36 Due to their apparent lack of expression of a tumorigenic phenotype in rodents,²⁵ MDCK cells have also been used to study neoplastic processes including epithelial-to-mesenchymal transition^23,27,28 and to assess the effects of viral oncogenes and chemical carcinogens on their phenotype.^13,32The results of studies that evaluate the ability of MDCK cells to form tumors in vivo have varied. Early studies found that these cells could produce tumors in chicken embryos but not in mature BALB/c nude mice.¹⁴ In contrast, MDCK cells formed progressively growing adenocarcinomas in newborn BALB/c nude mice, but tumor growth ceased as the pups approached maturity.²⁵ More recently, 2 different sublines of MDCK cells developed by independent groups were shown to be tumorigenic in athymic nude mice; but the incidence of tumor formation did not correlate with cell dose.^33-35As an initial approach to the study of neoplastic development in cells in culture, we evaluated the ability of MDCK cells to form tumors in athymic nude mice. We previously described the tumor-forming capacity of MDCK cells from different lots obtained from ATCC.²¹ That study revealed that MDCK cells from each of these lots formed tumors efficiently in adult and newborn nude mice, but the capacity of the cells to form tumors differed from lot to lot. During the initial experiments on MDCK cell tumor-forming efficiency in newborn nude mice, we observed what appeared to be a syndrome whose symptoms included tumor formation and disrupted growth leading to a failure-to-thrive (FTT) condition manifested by morbidity that required euthanasia of those pups most severely affected. During the study on the development of FTT, we found that the FTT syndrome occurred in newborn nude mice inoculated with 3 different sublines of MDCK cells. The current report describes an FTT syndrome associated with the formation of tumors by 10⁷ MDCK cells in newborn, athymic, nude mice.     

Phylogenetic analysis informed by geological history supports multiple, sequential invasions of the Mediterranean Basin by the angiosperm family Araceae

Despite the remarkable species richness of the Mediterranean flora and its well-known geological history, few studies have investigated its temporal and spatial origins. Most importantly, the relative contribution of geological processes and long-distance dispersal to the composition of contemporary Mediterranean biotas remains largely unknown. We used phylogenetic analyses of sequences from six chloroplast DNA markers, Bayesian dating methods, and ancestral area reconstructions, in combination with paleogeographic, paleoclimatic, and ecological evidence, to elucidate the time frame and biogeographic events associated with the diversification of Araceae in the Mediterranean Basin. We focused on the origin of four species, Ambrosina bassii, Biarum dispar, Helicodiceros muscivorus, Arum pictum, subendemic or endemic to Corsica, Sardinia, and the Balearic Archipelago. The results support two main invasions of the Mediterranean Basin by the Araceae, one from an area connecting North America and Eurasia in the Late Cretaceous and one from the Anatolian microplate in western Asia during the Late Eocene, thus confirming the proposed heterogeneous origins of the Mediterranean flora. The subendemic Ambrosina bassii and Biarum dispar likely diverged sympatrically from their widespread Mediterranean sister clades in the Early-Middle Eocene and Early-Middle Miocene, respectively. Combined evidence corroborates a relictual origin for the endemic Helicodiceros muscivorus and Arum pictum, the former apparently representing the first documented case of vicariance driven by the initial splitting of the Hercynian belt in the Early Oligocene. A recurrent theme emerging from our analyses is that land connections and interruptions, caused by repeated cycles of marine transgressions-regressions between the Tethys and Paratethys, favored geodispersalist expansion of biotic ranges from western Asia into the western Mediterranean Basin and subsequent allopatric speciation at different points in time from the Late Eocene to the Late Oligocene.