Trace elements in human parotid saliva

Although various proteins and some electrolytes have been measured in human saliva, little systematic data about the major and minor elemental components of this body fluid have been obtained. In order to obtain such data, concentrations of C, Na, P, Cl, K, Ca, Sc, Cr, Fe, Co, Zn, Se, Br, Rb, Sb, I, and Cs in human parotid saliva were measured by instrumental nuclear methods. The data obtained confirmed the relative lack of Zn in saliva of patients with hypogeusia (decreased taste acuity) and suggested that concentrations of Na, Cl, Br, and Ca followed the order: normals > hypogeusia > hyposmia (decreased smell acuity). To compare concentrations of elements in saliva with those in blood and urine, absolute concentrations were normalized to that of Na through the use of a concept called an enrichment factor. On this basis, parotid saliva is relatively depleted in Se, Zn, and Fe and enriched for most other elements relative to blood plasma indicating that the fluid is not simply a transudate of blood plasma. Using this same technique, saliva composition was found more similar to urine than blood plasma, being relatively depleted in Se, Cs, and Co, being enriched in I, Br, and Cr and having about the same relative concentrations of P, Cl, Zn, Fe, Ca, K, and Rb. As the total body concentrations of many of the enriched elements in saliva are extremely small, their enrichment in saliva suggests special roles for these elements in the oral cavity. Because of its accessibility, ease of collection, and interaction with some body constituents, saliva represents a useful, albeit neglected, tool in the diagnosis of some physiological and pathological changes in body function and in understanding important aspects of trace metal metabolism.     

Alpha7 Helix Plays an Important Role in the Conformational Stability of PTP1B

Abstract

The C-terminus of Protein Tyrosine Phosphatase 1B (PTP1B) includes an α-helix (α7), which forms an allosteric binding site 20 Å away from the active site. This helix is specific to PTP1B and its truncation decreases the catalytic activity significantly. Here, molecular dynamics (MD) simulations in the presence and absence of α7 were performed to investigate the role played by α7. The highly mobile α7 was found to maintain its contacts with loop 11 (L11)- α3 helix throughout the simulations. The interactions of Tyr152 on L11, Tyr176, Thr177 on the catalytically important WPD loop and Ser190 on α3 are important for the conformational stability and the concerted motions of the regions surrounding the WPD loop. In the absence of α7, L11 and WPD loop move away from their crystal structure conformations, resulting in the loss of the interactions in this region, and a decrease in the residue displacement correlations in the vicinity of WPD loop. Therefore, we suggest that one of the functionally important roles of α7 may be to limit the L11 and α3 motions, and, facilitate the WPD loop motions. Truncation of α7 in PTP1B is found to affect distant regions as well, such as the substrate recognition site and the phosphate binding-loop (P-loop), changing the conformations of these regions significantly. Our results show that the PTP1B specific α7 is important for the conformation and dynamics of the WPD loop, and also may play a role in ligand binding.     

Derivation of Human Induced Pluripotent Stem Cells from Patients with Maturity Onset Diabetes of the Young

Maturity onset diabetes of the young (MODY) is an autosomal dominant disease. Despite extensive research, the mechanism by which a mutant MODY gene results in monogenic diabetes is not yet clear due to the inaccessibility of patient samples. Induced pluripotency and directed differentiation toward the pancreatic lineage are now viable and attractive methods to uncover the molecular mechanisms underlying MODY. Here we report, for the first time, the derivation of human induced pluripotent stem cells (hiPSCs) from patients with five types of MODY: MODY1 (HNF4A), MODY2 (GCK), MODY3 (HNF1A), MODY5 (HNF1B), and MODY8 (CEL) with a polycistronic lentiviral vector expressing a Cre-excisable human “stem cell cassette” containing the four reprogramming factors OCT4, KLF4, SOX2, and CMYC. These MODY-hiPSCs morphologically resemble human pluripotent stem cells (hPSCs), express pluripotency markers OCT4, SOX2, NANOG, SSEA-4, and TRA-1–60, give rise to derivatives of the three germ layers in a teratoma assay, and are karyotypically normal. Overall, our MODY-hiPSCs serve as invaluable tools to dissect the role of MODY genes in the development of pancreas and islet cells and to evaluate their significance in regulating beta cell function. This knowledge will aid future attempts aimed at deriving functional mature beta cells from hPSCs.     

Molecular phylogeny of the Cricetinae subfamily based on the mitochondrial cytochrome b and 12S rRNA genes and the nuclear vWF gene

Despite some popularity of hamsters as pets and laboratory animals there is no reliable phylogeny of the subfamily Cricetinae available so far. Contradicting views exist not only about the actual number of species but also concerning the validity of several genera. We used partial DNA sequences of two mitochondrial (cytochrome b, 12S rRNA) and one partial nuclear gene (von Willebrand Factor exon 28) to provide a first gene tree of the Cricetinae based on 15 taxa comprising six genera. According to our data, Palaearctic hamsters fall into three distinct phylogenetic groups: Phodopus, Mesocricetus, and Cricetus-related species which evolved during the late Miocene about 7-12MY ago. Surprisingly, the genus Phodopus, which was previously thought to have appeared during the Pleistocene, forms the oldest clade. The largest number of extant hamster genera is found in a group of Cricetus-related hamsters. The genus Cricetulus itself proved to be not truly monophyletic with Cricetulus migratorius appearing more closely related to Tscherskia, Cricetus, and Allocricetulus. We propose to place the species within a new monotypic genus. Molecular clock calculations are not always in line with the dating of fossil records. DNA based divergence time estimates as well as taxonomic relationships demand a reevaluation of morphological characters previously used to identify fossils and extant hamsters.     

Alpha7 helix plays an important role in the conformational stability of PTP1B

The C-terminus of Protein Tyrosine Phosphatase 1B (PTP1B) includes an α-helix α7), which forms an allosteric binding site 20 ? away from the active site. This helix is specific to PTP1B and its truncation decreases the catalytic activity significantly. Here, molecular dynamics (MD) simulations in the presence and absence of α7 were performed to investigate the role played by α7. The highly mobile α7 was found to maintain its contacts with loop 11 (L11)α3 helix throughout the simulations. The interactions of Tyr152 on L11, Tyr176, Thr177 on the catalytically important WPD loop and Ser190 on α3 are important for the conformational stability and the concerted motions of the regions surrounding the WPD loop. In the absence of α7, L11 and WPD loop move away from their crystal structure conformations, resulting in the loss of the interactions in this region, and a decrease in the residue displacement correlations in the vicinity of WPD loop. Therefore, we suggest that one of the functionally important roles of α7 may be to limit the L11 and α3 motions, and, facilitate the WPD loop motions. Truncation of α7 in PTP1B is found to affect distant regions as well, such as the substrate recognition site and the phosphate binding-loop (P-loop), changing the conformations of these regions significantly. Our results show that the PTP1B specific α7 is important for the conformation and dynamics of the WPD loop, and also may play a role in ligand binding.     

Reactive oxygen species and ischemic cerebrovascular disease

Stroke is an emerging major health problem often resulting in death or disability. Hyperlipidemia, high blood pressure and diabetes are well established risk factors. Endothelial dysfunction associated with these risk factors underlies pathological processes leading to atherogenesis and cerebral ischemic injury. While mechanisms of disease are complex, endothelial dysfunction involves decreased nitric oxide (NO) and elevated levels of reactive oxygen species (ROS). At physiological levels, ROS participate in regulation of cellular metabolism. However, when ROS increase to toxic levels through imbalance of production and neutralization by antioxidant enzymes, they cause cellular injury in the form of lipid peroxidation, protein oxidation and DNA damage. Central nervous system cells are more vulnerable to ROS toxicity due to their inherent higher oxidative metabolism and less antioxidant enzymes, as well as higher content of membranous fatty acids. During ischemic stroke, ROS concentration rises from normal low levels to a peak point during reperfusion possibly underlying apoptosis or cellular necrosis. Clinical trials and animal studies have shown that natural compounds can reduce oxidative stress due to excessive ROS through their antioxidant properties. With further study, we may be able to incorporate these compounds into clinical use with potential efficacy for both the treatment and prevention of stroke.     

A Detailed Biological and Chemical Investigation of Sixteen Achillea Species’ Essential Oils via Chemometric Approach

Representatives of the Achillea genus are widely used as foods or nutraceuticals. Considering the increasing demand for herbal dietary supplements with health promoting effects, the objective of this research was to evaluate the chemical composition and biological activities of the essential oils obtained from sixteen Achillea species (A. biebersteinii, A. wilhelmsii subsp. wilhelmsii, A. aleppica subsp. zederbaueri, A. vermicularis, A. monocephala, A. nobilis, A. goniocephala, A. sintenisii, A. coarctata, A. kotschyi subsp. kotschyi, A. millefolium subsp. millefolium, A. lycaonica, A. spinulifolia, A. teretifolia, A. setacea, and A. schischkinii). Anticholinesterase, antiurease, antityrosinase enzymes inhibition, antioxidant, antimicrobial, toxic and cytotoxic activities of obtained essential oils were investigated. DPPH activities were found to be very low in all studied samples, while ABTS and CUPRAC antioxidant activities were found to be moderate. In addition, all samples were found to have moderate anticholinesterase and antimicrobial effects. It has been determined that the studied species have low cytotoxicity and high toxicity. Besides, chemical composition of the essential oils were determined by GC/MS and the results were chemometrically analyzed. The chemometric analyses of Achillea species collected from nine different regions were accomplished by principal component analysis (PCA) and hierarchical cluster analysis (HCA) techniques. According to the PCA analysis, A. nobilis subsp. neilreichii was found to be different from all studied species in terms of essential oil composition. The major components found in these species were piperitone, camphor, α‐terpinene, eucalyptol, artemisia ketone, endo‐borneol, β‐eudesmol and verbenol. The fact that camphor was toxic and found in majority of the studied species stands out as a remarkable result.     

De novo ChIP-seq analysis

Methods for the analysis of chromatin immunoprecipitation sequencing (ChIP-seq) data start by aligning the short reads to a reference genome. While often successful, they are not appropriate for cases where a reference genome is not available. Here we develop methods for de novo analysis of ChIP-seq data. Our methods combine de novo assembly with statistical tests enabling motif discovery without the use of a reference genome. We validate the performance of our method using human and mouse data. Analysis of fly data indicates that our method outperforms alignment based methods that utilize closely related species.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-015-0756-4) contains supplementary material, which is available to authorized users.     

Effects of protonation state of Asp181 and position of active site water molecules on the conformation of PTP1B

In protein tyrosine phosphatase 1B (PTP1B), the flexible WPD loop adopts a closed conformation (WPD_closed) in the active state of PTP1B, bringing the catalytic Asp181 close to the active site pocket, while WPD loop is in an open conformation (WPD_open) in the inactive state. Previous studies showed that Asp181 may be protonated at physiological pH, and ordered water molecules exist in the active site. In the current study, molecular dynamics simulations are employed at different Asp181 protonation states and initial positions of active site water molecules, and compared with the existing crystallographic data of PTP1B. In WPD_closed conformation, the active site is found to maintain its conformation only in the protonated state of Asp181 in both free and liganded states, while Asp181 is likely to be deprotonated in WPD_open conformation. When the active site water molecule network that is a part of the free WPD_closed crystal structure is disrupted, intermediate WPD loop conformations, similar to that in the PTPRR crystal structure, are sampled in the MD simulations. In liganded PTP1B, one active site water molecule is found to be important for facilitating the orientation of Cys215 and the phosphate ion, thus may play a role in the reaction. In conclusion, conformational stability of WPD loop, and possibly catalytic activity of PTP1B, is significantly affected by the protonation state of Asp181 and position of active site water molecules, showing that these aspects should be taken into consideration both in MD simulations and inhibitor design. © Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Effects of caffeic acid phenethyl ester on matrix molecules and angiogenetic and anti-angiogenetic factors in gastric cancer cells cultured on different substrates

Migration, invasion, metastasis and angiogenesis associated with cancer depend on the surrounding microenvironment. Angiogenesis, the growth of new capillaries, is a regulator of cancer growth and a useful target for cancer therapy. We examined matrix protein interactions in a gastric cancer cell culture that was treated with different doses of caffeic acid (3,4-dihydroxycinnamic acid) phenethyl ester (CAPE). We also investigated the relations among the levels of vascular endothelial growth factor (VEGF), matrix metalloproteinase-9 (MMP-9), endostatin (ES) and trombospondin-1 (TSP-1). Cytotoxity of CAPE was measured using the 3-(4,5-dmethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. We examined the behavior of cells on laminin and collagen I coated surfaces in response to the angiogenic effect of these matrix molecules. We examined the protein alterations of these matrix molecules immunohistochemically and measured the levels of VEGF, MMP-9, ES and TSP-1 using the ELISA test. We showed that application of CAPE to the gastric cancer cell line on tissue culture plastic, laminin and collagen I significantly decreased the VEGF and MMP-9 protein levels. We found that TSP-1 levels were increased significantly in the gastric cancer cells after application of CAPE. The protein levels of gastric cancer cells also were increased significantly when tissue was cultured on laminin and collagen I. Application of CAPE to cells on laminin or collagen I coated surfaces significantly increased all of the proteins except ES. ES levels were increased on the collagen I covered surfaces, but the laminin surface decreased the levels of ES significantly. We demonstrated the beneficial effect of CAPE on a gastric cancer cell line including inhibition of proliferation and induction of some proteins that might be related to decreased angiogenesis.