Integration potential of mouse and human bone marrow-derived mesenchymal stem cells

Mesenchymal stem cells (MSCs) are a multipotent cell population which has been described to exert renoprotective and regenerative effects in experimental models of kidney injury. Several lines of evidence indicate that MSCs also have the ability to contribute to nephrogenesis, suggesting that the cells can be employed in stem cell-based applications aimed at de novo renal tissue generation. In this study we re-evaluate the capacity of mouse and human bone marrow-derived MSCs to contribute to the development of renal tissue using a novel method of embryonic kidney culture. Although MSCs show expression of some genes involved in renal development, their contribution to nephrogenesis is very limited in comparison to other stem cell types tested. Furthermore, we found that both mouse and human MSCs have a detrimental effect on the ex vivo development of mouse embryonic kidney, this effect being mediated through a paracrine action. Stimulation with conditioned medium from a mouse renal progenitor population increases the ability of mouse MSCs to integrate into developing renal tissue and prevents the negative effects on kidney development, but does not appear to enhance their ability to undergo nephrogenesis.     

MTHFR C677T polymorphism in chronic pancreatitis and pancreatic adenocarcinoma

Chronic pancreatitis and pancreatic adenocarcinoma are extensively studied as common and potentially lethal disorders. However, their causes and genetic background in most cases remain unclear. The C677T polymorphism in 5',10'-methylenetetrahydrofolate reductase (MTHFR) gene may modulate the risk of pancreatic disorders. In this study, we tested whether MTHFR C677T polymorphism is associated with chronic pancreatitis and pancreatic adenocarcinoma in the Serbian population. DNA was extracted from blood samples of 51 chronic pancreatitis patients, 21 pancreatic adenocarcinoma patients, and a control group consisting of 50 healthy smokers. The MTHFR C677T polymorphism was analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. Although, no statistically significant differences were observed in the distribution of MTHFR genotype or allele frequencies between patients and control groups, the results showed an increased frequency of homozygotes for MTHFR C677T polymorphism in chronic pancreatitis patients (14%) and a decreased frequency in pancreatic adenocarcinoma patients (5%) in comparison to the control group (8%). We speculate that the MTHFR C677T polymorphism could act as a possible risk factor for chronic pancreatitis and a possible protective factor in pancreatic adenocarcinoma. This observation needs further investigation in prospective studies on a larger number of patients, in which the effect of other genetic and environmental factors should also be taken into consideration.     

Ecm11 protein of yeast Saccharomyces cerevisiae is regulated by sumoylation during meiosis

Damaged regulation of the small ubiquitin-like modifier (SUMO) system contributes to some human diseases; therefore, it is very important to identify the SUMO targets and to determine the function of their sumoylation. In this study, it is shown that Ecm11 protein in Saccharomyces cerevisiae is modified by SUMO during meiosis. It is known that Ecm11 is required in the early stages of yeast meiosis where its function is related to DNA replication and crossing over. Here it is shown that the level of Ecm11 protein is low in mitosis, but high in meiosis. The highest level of Ecm11 is in the early-middle phase of sporulation. A specific site of sumoylation was identified in Ecm11 at Lys5 and evidence is provided that sumoylation at this site directly regulates Ecm11 function in meiosis. On the other hand, no relationship was observed between sumoylation of Ecm11 and its role during vegetative growth. It was shown that Ecm11 interacts with Siz2 SUMO ligase in a two-hybrid system; although Siz2 is not essential for the Ecm11 sumoylation.     

Quantitative phosphoproteomics by mass spectrometry: past, present, and future

Protein phosphorylation-mediated signaling networks regulate much of the cellular response to external stimuli, and dysregulation in these networks has been linked to multiple disease states. Significant advancements have been made over the past decade to enable the analysis and quantification of cellular protein phosphorylation events, but comprehensive analysis of the phosphoproteome is still lacking, as is the ability to monitor signaling at the network level while comprehending the biological implications of each phosphorylation site. In this review we highlight many of the technological advances over the past decade and describe some of the latest applications of these tools to uncover signaling networks in a variety of biological settings. We finish with a concise discussion of the future of the field, including additional advances that are required to link protein phosphorylation analysis with biological insight.     

Identification of Ruta graveolens L. metabolites accumulated in the presence of abiotic elicitors

The study aimed to elucidate the effects of benzothiadiazole (BTH) and saccharin on the biosynthesis of simple coumarins, linear furanocoumarins, dihydrofuranocoumarins, and furoquinolone alkaloids in shoots of R. graveolens cultivated in vitro. The biosynthesized metabolites were analyzed and identified by GC-MS and by comparison of Kovats indices. Eight coumarin metabolites were identified: bergapten, chalepin, isopimpinelin, pinnarin, psoralen, rutacultin, rutamarin, and xanthotoxin, and also four alkaloids: dictamnine, gamma-fagarine, skimmianine, and kokusaginine. Each of the tested BTH concentrations induced a significant production of furanocoumarins and furoquinolone alkaloids. The use of saccharin also increased the production of bergapten, isopimpinelin, pinnarin, psoralen, and xanthotoxin several times.     

Complex I within oxidatively stressed bovine heart mitochondria is glutathionylated on Cys-531 and Cys-704 of the 75-kDa subunit: potential role of CYS residues in decreasing oxidative damage

Complex I has reactive thiols on its surface that interact with the mitochondrial glutathione pool and are implicated in oxidative damage in many pathologies. However, the Cys residues and the thiol modifications involved are not known. Here we investigate complex I thiol modification within oxidatively stressed mammalian mitochondria, containing physiological levels of glutathione and glutaredoxin 2. In mitochondria incubated with the thiol oxidant diamide, complex I is only glutathionylated on the 75-kDa subunit. Of the 17 Cys residues on the 75-kDa subunit, 6 are not involved in iron-sulfur centers, making them plausible candidates for glutathionylation. Mass spectrometry of complex I from oxidatively stressed bovine heart mitochondria showed that only Cys-531 and Cys-704 were glutathionylated. The other four non-iron-sulfur center Cys residues remained as free thiols. Complex I glutathionylation also occurred in response to relatively mild oxidative stress caused by increased superoxide production from the respiratory chain. Although complex I glutathionylation within oxidatively stressed mitochondria correlated with loss of activity, it did not increase superoxide formation, and reversal of glutathionylation did not restore complex I activity. Comparison with the known structure of the 75-kDa ortholog Nqo3 from Thermus thermophilus complex I suggested that Cys-531 and Cys-704 are on the surface of mammalian complex I, exposed to the mitochondrial glutathione pool. These findings suggest that Cys-531 and Cys-704 may be important in preventing oxidative damage to complex I by reacting with free radicals and other damaging species, with subsequent glutathionylation recycling the thiyl radicals and sulfenic acids formed on the Cys residues back to free thiols.     

MHC and Preferences for Male Odour in the Bank Vole

Highly polymorphic major histocompatibility complex (MHC) genes are thought to play a central role in the choice of genetically compatible sexual partners in some vertebrates, although the evidence is variable across species. Here, we investigate the association between similarity in the MHC region and sexual preferences in the bank vole Myodes ( =Clethrionomys ) glareolus (Arvicollinae) in a laboratory setting. Females in post-partum oestrus were given the choice between the scents of two males in a Y-maze. Both males were unrelated to the female, but differed in their MHC similarity to the female. We found that females spent more time near the scent of MHC dissimilar males than those, with whom they shared MHC alleles. This suggests that bank voles use MHC-related cues to choose compatible mates.     

Formation of noncanonical DNA structures mediated by human ORC4, a protein component of the origin recognition complex

Many genomic sequences, DNA replication origins included, contain specific structural motifs prone to alternative base pairing. Structural rearrangements of DNA require specific environmental conditions and could be favored by chemical agents or proteins. To improve our understanding of alternative conformations of origins and the manner in which they form, we have investigated the effect of DNA-binding, AAA+ protein human ORC4 on single-stranded origin DNA or various oligonucleotides. Here we demonstrate that human ORC4 stimulated formation of inter- and intramolecular T.A.T triplexes and created novel structures, such as homoadenine duplexes. Adenine-based structures were held together by Hoogsteen hydrogen bonds, as demonstrated on 7-deaza-dAMP- or dAMP-containing substrates, and characterized by increased thermal stability. Adenine pairing occurred only in the presence of human ORC4, in a neutral buffer supplemented with ATP and Mg (2+) ions. The protein mutant that could not bind ATP was inactive in this reaction. Since the action of human ORC4 could be biologically important, its potential impact on DNA replication is discussed.     

The Differential Killing of Genes by Inversions in Prokaryotic Genomes

We have elaborated a method which has allowed us to estimate the direction of translocation of orthologs which have changed, during the phylogeny, their positions on chromosome in respect to the leading or lagging role of DNA strands. We have shown that the relative number of translocations which have switched positions of genes from the leading to the lagging DNA strand is lower than the number of translocations which have transferred genes from the lagging strand to the leading strand of prokaryotic genomes. This paradox could be explained by assuming that the stronger mutation pressure and selection after inversion preferentially eliminate genes transferred from the leading to the lagging DNA strand. Received: 12 December 2000 / Accepted: 20 April 2001