Telomeres as age markers in vertebrate molecular ecology

Chronological age is a fundamental and yet elusive variable in studies of many wild animals. Telomeres are nucleoprotein structures on the ends of chromosomes that change size throughout the life of many animals and because of this property have been advocated as a means to estimate age. In this review, we assess the existing and potential application of using telomeres for age estimation. We argue that there are conceptual and statistical inconsistencies in previous studies and that the basis for telomere change over time is not well understood and affected by several intrinsic and extrinsic process unrelated to chronological time. Furthermore, these processes are likely to vary spatially and temporally for animal populations. We conclude that the current data suggest telomeres should not be used for age estimation. If telomere-based age estimation is to be used, more work in understanding variability in key processes affecting telomere dynamics and rigorous substantiation via blind testing is needed.     

Proinflammatory factors in saliva as possible markers for periodontal disease

Studies have indicated that host inflammatory proteins, enzymes and indicators of bone metabolism present in saliva differ in different types of periodontal disease. However, the number of markers analyzed was limited and the effect of edentulousness was not examined. We measured the concentration of host inflammatory proteins: C-reactive protein (CRP), C3 and C4 complement components, alpha-2-macroglobulin (alpha-2M) and tumor-necrosis factor (TNF) in unstimulated saliva of 14 periodontally healthy (PH), 9 edentulous persons (EP), 10 patients with chronic periodontitis (CP) and 18 with aggressive periodontitis (AgP). TNF was below the level of detection in all samples except one. Edentulous persons and patients with CP had significantly reduced concentrations of CRP, C3 and alpha-2M. Edentulous persons and AgP patients had lower C4 concentrations. We can conclude that edentulous persons and CP patients have reduced salivary concentrations of host inflammatory proteins. These findings suggest that a reduction in host responsiveness might play a role in the pathogenesis of CP.     

Trypsin-like proteins of the fungi as possible markers of pathogenicity

Sequences of peptidases with conserved motifs around the active site residues that are characteristic of trypsins (similar to trypsin peptidases, STP) were obtained from publicly-available fungal genomes and related databases. Among the 75 fungal genomes, 29 species of parasitic Ascomycota contained genes encoding STP and their homologs. Searches of non-redundant protein sequences, patented protein sequences, and expressed sequence tags resulted in another 18 STP sequences in 10 fungal species from Ascomycota, Basidiomycota, and Zygomycota. A comparison of fungi species containing STP sequences revealed that almost all are pathogens of plants, animals or fungi. A comparison of the primary structure of homologous proteins, including the residues responsible for substrate binding and specificity of the enzyme, revealed three groups of homologous sequences, all presumably from S1 family: trypsin-like peptidases, chymotrypsin-like peptidases and serine peptidases with unknown substrate specificity. Homologs that are presumably functionally inactive were predicted in all groups. The results in general support the hypothesis that the expression of trypsin-like peptidases in fungi represents a marker of fungal phytopathogenicity. A phylogenetic tree was constructed using peptidase and homolog amino acid sequences, demonstrating that all have noticeable differences and almost immediately deviate from the common root. Therefore, we conclude that the changes that occurred in STP of pathogenic fungi in the course of evolution represent specific adaptations to proteins of their respective hosts, and mutations in peptidase genes are important components of life-style changes and taxonomic divergence.     

Arabinogalactan proteins as molecular markers in Arabidopsis thaliana sexual reproduction

Some of the most important changes that occur in plants during sexual reproduction involve the transition from a sporophytic to a gametophytic type of development. In this paper, these changes were evaluated for Arabidopsis thaliana. The results obtained clearly show differences in the pattern of distribution of specific arabinogalactan protein (AGP) sugar epitopes, during anther and ovule development. AGPs are hydroxyproline-rich glycoproteins that are massively glycosylated and ubiquitous in plants. The molecular mechanism of action of AGPs is still unknown, mainly due to the difficulties posed by the complex saccharide chains. However, the complex structure of the sugar fraction of AGPs makes them a potential source of signalling molecules. The selective labelling obtained with AGP mAbs JIM8, JIM13, MAC207, and LM2, during Arabidopsis pollen and pistil development, suggests that some AGPs can work as markers for gametophytic cell differentiation. Specific labelling of the first gametophytic cells in the pistil, the strong labelling of the secretory cells of the embryo sac, the synergid cells, and the labelling of the integument micropylar cells, apparently outlining the pollen tube pathway into its final target, the embryo sac, have all been shown. In the anthers, the specific labelling of gametophytic cells, and of the male gametes that travel along the pollen tube, may indicate AGP epitopes acting as signals for the pollen tube to reach its final destiny. The specific labelling of cells destined to go into programmed cell death is also discussed.     

Identification of phosphoproteins as possible differentiation markers in all-trans-retinoic acid-treated neuroblastoma cells

Background

Neuroblastic tumors account for 9–10% of pediatric tumors and neuroblastoma (NB) is the first cause of death in pre-school age children. NB is classified in four stages, depending on the extent of spreading. A fifth type of NB, so-called stage 4S (S for special), includes patients with metastatic tumors but with an overall survival that approximates 75% at five years. In most of these cases, the tumor regresses spontaneously and regression is probably associated with delayed neuroblast cell differentiation.

Methodology/Principal Findings

In order to identify new early markers to follow and predict this process for diagnostic and therapeutics intents, we mimicked the differentiation process treating NB cell line SJ-NK-P with all-trans-retinoic acid (ATRA) at different times; therefore the cell proteomic pattern by mass spectrometry and the phosphoproteomic pattern by a 2-DE approach coupled with anti-phosphoserine and anti-phosphotyrosine western blotting were studied.

Conclusions/Significance

Proteomic analysis identified only two proteins whose expression was significantly different in treated cells versus control cells: nucleoside diphosphate kinase A (NDKA) and reticulocalbin-1 (RCN1), which were both downregulated after 9 days of ATRA treatment. However, phosphoproteomic analysis identified 8 proteins that were differentially serine-phosphorylated and 3 that were differentially tyrosine-phosphorylated after ATRA treatment. All proteins were significantly regulated (at least 0.5-fold down-regulated). Our results suggest that differentially phosphorylated proteins could be considered as more promising markers of differentiation for NB than differentially expressed proteins.     

An investigation of seven enzymes as possible genetic markers in horse leucocytes

In this paper we describe seven enzymes, NP, GOT_M, PGM₂, αFUC, PEP A, ADA and MPI which are found in the white cells of horses, including 39 British crossbred ponies and 16 crossbred horses, 30 Mongolian ponies and 10 Icelandic ponies. Two of these enzymes - αFUC and MPI - were polymorphic in all the populations of horses studied and could prove useful as additional markers in the paternity testing of horses. PEP A and GOT_M were also polymorphic in two of the populations studied and could be used as further markers in these populations.     

Evidence for mammalian collagenases as zinc ion metalloenzymes.

Collagenases (EC 3.4.24.3) from human skin, rat skin and rat uterus were inhibited by the chelating agents EDTA, 1,10-phenanthroline and tetraethylene pentamine in the presence of excess Ca2+, suggesting that a second metal ion participates in the activity of the enzyme. Collagenase inhibition by 1,10-phenanthroline could be both prevented and reversed by a number of transition metal ions, specifically Zn2+, Co2+, Fe2+ and Cu2+. However, Zn2+ is effective in five-fold lower molar concentrations (1-10(-4) M) than the other ions. Furthermore, Zn2+ was the only ion tested able to prevent and reverse the inhibition of collagenase by EDTA in the presence of excess Ca2+. Atomic absorption analysis of purified collagenase for Zn2+ showed that Zn2+ was present in the enzyme preparations, and that the metal co-purifies with collagenase during column chromatography.     

Species-specific oocyte proteins as molecular markers for Lineus taxonomy

The yolk proteins from the eggs of five species of thenemertean genus Lineus were analysed and theresulting data were used to define L. longissimus, L. sanguineus, L. lacteus, L. ruber, and L. viridis as distinct taxa (species to date). All fivespecies have at least one large (80–60 kDa) and onemedium sized (45–30 kDa) vitellin subunit, but thereare significant differences in the number and sizes ofthe molecular subunits. The large and medium sizedvitellin components of all the species areglycosylated, except for the 145 kDa protein of L. sanguineus. Rabbit antibodies to the L. lacteus vitellin subunits cross-reacted withthe vitellin subunits in the eggs of the otherspecies. The vitellin components of these five speciesof nemertean are very similar. Two species, L.ruber and L. viridis, lay their eggsin gelatinous masses, and the electrophoretic patternsof the jelly proteins show that the physicalconsistency of the jelly depends on molecular weightsof the components. The egg mass of L. viridis contains smaller proteins than the egg massof L. ruber. The repeatablespecies-specific patterns of vitellin componentsprovide a useful complement to the usual taxonomiccriteria.     

Species-specific oocyte proteins as molecular markers for Lineus taxonomy

The yolk proteins from the eggs of five species of thenemertean genus Lineus were analysed and theresulting data were used to define L. longissimus, L. sanguineus, L. lacteus, L. ruber, and L. viridis as distinct taxa (species to date). All fivespecies have at least one large (80–60 kDa) and onemedium sized (45–30 kDa) vitellin subunit, but thereare significant differences in the number and sizes ofthe molecular subunits. The large and medium sizedvitellin components of all the species areglycosylated, except for the 145 kDa protein of L. sanguineus. Rabbit antibodies to the L. lacteus vitellin subunits cross-reacted withthe vitellin subunits in the eggs of the otherspecies. The vitellin components of these five speciesof nemertean are very similar. Two species, L.ruber and L. viridis, lay their eggsin gelatinous masses, and the electrophoretic patternsof the jelly proteins show that the physicalconsistency of the jelly depends on molecular weightsof the components. The egg mass of L. viridis contains smaller proteins than the egg massof L. ruber. The repeatablespecies-specific patterns of vitellin componentsprovide a useful complement to the usual taxonomiccriteria.     

Fibrinogen alpha chain O-glycopeptides as possible markers of urinary tract infection

Urinary tract infection (UTI) is the most common bacterial infection leading to substantial morbidity and considerable health care expenditures across all ages. Here we present an exploratory UPLC-MS study of human urine in the context of febrile, complicated urinary tract infection aimed to reveal and identify possible markers of a host response on infection. A UPLC-MS based workflow, taking advantage of Ultra High Resolution (UHR) Qq-ToF-MS, and multivariate data handling were applied to a carefully selected group of 39 subjects with culture-confirmed febrile Escherichia coli UTI. Using a combination of unsupervised and supervised multivariate modeling we have pinpointed a number of peptides specific for UTI. An unequivocal structural identification of these peptides, as O-glycosylated fragments of the human fibrinogen alpha 1 chain, required MS2 and MS3 experiments on two different MS platforms: ESI-UHR-Qq-ToF and ESI-ion trap, a blast search and, finally, confirmation was achieved by matching experimental tandem mass spectra with those of custom synthesized candidate-peptides.In conclusion, exploiting non-targeted UPLC-MS based approach for the investigation of UTI related changes in urine, we have identified and structurally characterized unique O-glycopeptides, which are, to our knowledge, the first demonstration of O-glycosylation of human fibrinogen alpha 1-chain.     

An investigation of seven enzymes as possible genetic markers in horse leucocytes.

In this paper we describe seven enzymes, NP, GOTM, PGM2, alpha FUC, PEP A, ADA and MPI which are found in the white cells of horses, including 39 British crossbred ponies and 16 crossbred horses, 30 Mongolian ponies and 10 Icelandic ponies. Two of these enzymes--alpha FUC and MPI--were polymorphic in all the populations of horses studied and could prove useful as additional markers in the paternity testing of horses. PEP A and GOTM were also polymorphic in two of the populations studied and could be used as further markers in these populations.     

Surprising cofactors in metalloenzymes

Transition metal complexes are located at the active sites of a number of enzymes involved in intriguing biochemical reactions. These complexes can exhibit a wide variety of chemical reactivity due to the ease at which transition metals can adopt different coordination environments and oxidation states. Crystallography has been a powerful technique for examining the structure and conformational variability of complex biological metallocenters. In particular, the past ten years have provided a wealth of structural information and several surprises concerning the metallocenters at the active sites of nitrogenase, hydrogenase and carbon monoxide dehydrogenase/acetyl-coenzyme A synthase.     

New partial sequences of phosphoenolpyruvate carboxylase as molecular phylogenetic markers

To better understand the evolution of the enzyme phosphoenolpyruvate carboxylase (PEPC) and to test its versatility as a molecular character in phylogenetic and taxonomic studies, we have characterized and compared 70 new partial PEPC nucleotide and amino acid sequences (about 1100 bp of the 3' side of the gene) from 50 plant species (24 species of Bryophyta, 1 of Pteridophyta, and 25 of Spermatophyta). Together with previously published data, the new set of sequences allowed us to construct the up to now most complete phylogenetic tree of PEPC, where the PEPC sequences cluster according to both the taxonomic positions of the donor plants and the assumed specific function of the PEPC isoforms. Altogether, the study further strengthens the view that PEPC sequences can provide interesting information for the reconstruction of phylogenetic relations between organisms and metabolic pathways. To avoid confusion in future discussion, we propose a new nomenclature for the denotation of PEPC isoforms.     

Application of two microsatellite sequences in wheat storage proteins as molecular markers

In eukaryotes, tandem arrays of simple-sequence repeat sequences can find applications as highly variable and multi-allelic PCR-based genetic markers. In hexaploid bread wheat, a large-genome inbreeding species with low levels of RFLP, di- and trinucleotide tandem repeats were found in 22 published gene sequences, two of which were converted to PCR-based markers. These were shown to be genome-specific and displayed high levels of variation. These characteristics make them especially suitable for intervarietal breeding applications.