Salivary protein changes in response to acute stress in medical residents performing advanced clinical simulations: a pilot proteomics study

Context: Quantitative changes of salivary proteins due to acute stress were detected.

Objective: To explore protein markers of stress in saliva of eight medical residents who performed emergency medicine simulations.

Materials and methods: Saliva was collected before the simulations, after the simulations, and following morning upon waking. Proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), identified by mass spectrometry (MS), and relatively quantified by densitometry.

Results: Salivary alpha-amylase and S–type cystatins significantly increased, while the ～26?kDa and low-molecular weight (MW) (<10?kDa) SDS-PAGE bands exhibited changes after stress.

Discussion and conclusion: Alpha-amylase and cystatins are potential salivary markers of acute stress, but further validation should be performed using larger sample populations.     

Mass spectrometric and mutational analyses reveal Lys-6-linked polyubiquitin chains catalyzed by BRCA1-BARD1 ubiquitin ligase

The breast and ovarian cancer suppressor BRCA1 acquires significant ubiquitin ligase activity when bound to BARD1 as a RING heterodimer. Although the activity may well be important for the role of BRCA1 as a tumor suppressor, the biochemical consequence of the activity is not yet known. Here we report that BRCA1-BARD1 catalyzes Lys-6-linked polyubiquitin chain formation. K6R mutation of ubiquitin dramatically reduces the polyubiquitin products mediated by BRCA1-BARD1 in vitro. BRCA1-BARD1 preferentially utilizes ubiquitin with a single Lys residue at Lys-6 or Lys-29 to mediate autoubiquitination of BRCA1 in vivo. Furthermore, mass spectrometry analysis identified the Lys-6-linked branched ubiquitin fragment from the polyubiquitin chain produced by BRCA1-BARD1 using wild type ubiquitin. The BRCA1-BARD1-mediated Lys-6-linked polyubiquitin chains are deubiquitinated by 26 S proteasome in vitro, whereas autoubiquitinated CUL1 through Lys-48-linked polyubiquitin chains is degraded. Proteasome inhibitors do not alter the steady state level of the autoubiquitinated BRCA1 in vivo. Hence, the results indicate that BRCA1-BARD1 mediates novel polyubiquitin chains that may be distinctly edited by 26 S proteasome from conventional Lys-48-linked polyubiquitin chains.     

LOCOMOTOR PERFORMANCE AT LOW TEMPERATURE AND THE EVOLUTION OF NOCTURNALITY IN GECKOS

Nocturnal geckos are active at body temperatures 10–35°C below the thermal optima for maximum rate of aerobic metabolism of diurnal lizards. Therefore, given ancestral (diurnal) lizard physiology, nocturnality causes a substantial thermal handicap in locomotor performance. In prior studies, we hypothesized that a low minimum cost of locomotion (C_min) in geckos was an adaptation that increased locomotor endurance capacity at low, nocturnal temperatures. However, C_min is only part of an integrated system that, in conjunction with the maximum rate of oxygen consumption, sets the maximum speed that can be sustained aerobically (termed the maximum aerobic speed or MAS). We conducted the first phylogenetic analysis of MAS and lizards and found that the greatest changes in MAS, C_min and (at activity temperatures) in the evolutionary history of lizards all coincided with the evolution of nocturnality in geckos. Geckos active at 15–25°C did not become optimized for nocturnal temperatures, or fully offset the thermal effects of nocturnality by evolving maximal rates of oxygen consumption comparable to diurnal lizards active at 35°C. Geckos did evolve MAS twice that of diurnal lizards running at low temperatures by evolving a remarkably low C_min. Allometric analysis and phylogenetically independent contrasts of , C_min, and MAS indicate a 72% evolutionary decrease in , (at activity temperatures) and a 50% evolutionary decrease in C_min concordant with the evolution of nocturnality in geckos. Experimental measurements show that decreased C_min in six species of gecko increased MAS by 50–120% compared to diurnal lizards at low temperatures. Thus, geckos sufficiently overcame the near paralyzing effects of nocturnal temperatures, but only offset about 50% of the decrease in MAS resulting from the low maximum rate of oxygen consumption. Although the nocturnal environment remains severely suboptimal, the evolution of a low cost of locomotion in the ancestor of geckos was highly adaptive for nocturnality. We also present a generalized approach to ecophysiological evolution that integrates phylogeny with the causal relationships among environment, physiology, and performance capacity. With respect to a clade, two hypotheses are central to our integrative approach: (1) a change of an environmental variable (e.g., temperature) causes a performance handicap; and (2) evolution of a physiological variable (e.g., minimum cost of locomotion [C_min]) increases performance in the derived environment. To test the hypothesis that evolution of a physiological variable is adaptive in nature, we suggest determining if individuals in nature perform at levels exceeding the performance capacity of their hypothetical ancestors and if this additional performance capacity is due to the evolution of the physiological variable in question.     

Targeted tandem affinity purification of PSD‐95 recovers core postsynaptic complexes and schizophrenia susceptibility proteins

The molecular complexity of mammalian proteomes demands new methods for mapping the organization of multiprotein complexes. Here, we combine mouse genetics and proteomics to characterize synapse protein complexes and interaction networks. New tandem affinity purification (TAP) tags were fused to the carboxyl terminus of PSD‐95 using gene targeting in mice. Homozygous mice showed no detectable abnormalities in PSD‐95 expression, subcellular localization or synaptic electrophysiological function. Analysis of multiprotein complexes purified under native conditions by mass spectrometry defined known and new interactors: 118 proteins comprising crucial functional components of synapses, including glutamate receptors, K⁺ channels, scaffolding and signaling proteins, were recovered. Network clustering of protein interactions generated five connected clusters, with two clusters containing all the major ionotropic glutamate receptors and one cluster with voltage‐dependent K⁺ channels. Annotation of clusters with human disease associations revealed that multiple disorders map to the network, with a significant correlation of schizophrenia within the glutamate receptor clusters. This targeted TAP tagging strategy is generally applicable to mammalian proteomics and systems biology approaches to disease.     

Absence of DICER in Monocytes and Its Regulation by HIV-1

MicroRNAs (miRNAs) are a class of small RNA molecules that function to control gene expression and restrict viral replication in host cells. The production of miRNAs is believed to be dependent upon the DICER enzyme. Available evidence suggests that in T lymphocytes, HIV-1 can both suppress and co-opt the host''s miRNA pathway for its own benefit. In this study, we examined the state of miRNA production in monocytes and macrophages as well as the consequences of viral infection upon the production of miRNA. Monocytes in general express low amounts of miRNA-related proteins, and DICER in particular could not be detected until after monocytes were differentiated into macrophages. In the case where HIV-1 was present prior to differentiation, the expression of DICER was suppressed. MicroRNA chip results for RNA isolated from transfected and treated cells indicated that a drop in miRNA production coincided with DICER protein suppression in macrophages. We found that the expression of DICER in monocytes is restricted by miR-106a, but HIV-1 suppressed DICER expression via the viral gene Vpr. Additionally, analysis of miRNA expression in monocytes and macrophages revealed evidence that some miRNAs can be processed by both DICER and PIWIL4. Results presented here have implications for both the pathology of viral infections in macrophages and the biogenesis of miRNAs. First, HIV-1 suppresses the expression and function of DICER in macrophages via a previously unknown mechanism. Second, the presence of miRNAs in monocytes lacking DICER indicates that some miRNAs can be generated by proteins other than DICER.     

Controlling tick-borne diseases through domestic animal management: a theoretical approach

Vector-borne diseases are of global importance to human and animal health. Empirical trials of effective methods to control vectors and their pathogens can be difficult for practical, financial and ethical reasons. Here, therefore, we use a mathematical model to predict the effectiveness of a vector-borne disease control method. As a case study, we use the tick-louping ill virus system, where sheep are treated with acaricide in an attempt to control ticks and disease in red grouse , an economically important game bird. We ran the model under different scenarios of sheep flock sizes, alternative host (deer) densities, acaricide efficacies and tick burdens. The model predicted that, with very low deer densities, using sheep as tick mops can reduce the tick population and virus prevalence. However, treatment is ineffective above a certain threshold deer density, dependent on the comparative tick burden on sheep and deer. The model also predicted that high efficacy levels of acaricide must be maintained for effective tick control. This study suggests that benignly managing one host species to protect another host species from a vector and pathogen can be effective under certain conditions. It also highlights the importance of understanding the ecological complexity of a system, in order to target control methods only under certain circumstances for maximum effectiveness.     

Recent amplification of the kangaroo endogenous retrovirus, KERV, limited to the centromere

Mammalian retrotransposons, transposable elements that are processed through an RNA intermediate, are categorized as short interspersed elements (SINEs), long interspersed elements (LINEs), and long terminal repeat (LTR) retroelements, which include endogenous retroviruses. The ability of transposable elements to autonomously amplify led to their initial characterization as selfish or junk DNA; however, it is now known that they may acquire specific cellular functions in a genome and are implicated in host defense mechanisms as well as in genome evolution. Interactions between classes of transposable elements may exert a markedly different and potentially more significant effect on a genome than interactions between members of a single class of transposable elements. We examined the genomic structure and evolution of the kangaroo endogenous retrovirus (KERV) in the marsupial genus Macropus. The complete proviral structure of the kangaroo endogenous retrovirus, phylogenetic relationship among relative retroviruses, and expression of this virus in both Macropus rufogriseus and M. eugenii are presented for the first time. In addition, we show the relative copy number and distribution of the kangaroo endogenous retrovirus in the Macropus genus. Our data indicate that amplification of the kangaroo endogenous retrovirus occurred in a lineage-specific fashion, is restricted to the centromeres, and is not correlated with LINE depletion. Finally, analysis of KERV long terminal repeat sequences using massively parallel sequencing indicates that the recent amplification in M. rufogriseus is likely due to duplications and concerted evolution rather than a high number of independent insertion events.     

Large-scale identification of proteins in human salivary proteome by liquid chromatography/mass spectrometry and two-dimensional gel electrophoresis-mass spectrometry

Human saliva contains a large number of proteins and peptides (salivary proteome) that help maintain homeostasis in the oral cavity. Global analysis of human salivary proteome is important for understanding oral health and disease pathogenesis. In this study, large-scale identification of salivary proteins was demonstrated by using shotgun proteomics and two-dimensinal gel electrophoresis-mass spectrometry (2-DE-MS). For the shotgun approach, whole saliva proteins were prefractionated according to molecular weight. The smallest fraction, presumably containing salivary peptides, was directly separated by capillary liquid chromatography (LC). However, the large protein fractions were digested into peptides for subsequent LC separation. Separated peptides were analyzed by on-line electrospray tandem mass spectrometry (MS/MS) using a quadrupole-time of flight mass spectrometer, and the obtained spectra were automatically processed to search human protein sequence database for protein identification. Additionally, 2-DE was used to map out the proteins in whole saliva. Protein spots 105 in number were excised and in-gel digested; and the resulting peptide fragments were measured by matrix-assisted laser desorption/ionization-mass spectrometry and sequenced by LC-MS/MS for protein identification. In total, we cataloged 309 proteins from human whole saliva by using these two proteomic approaches.     

Escherichia coli aceE variants coding pyruvate dehydrogenase improve the generation of pyruvate‐derived acetoin

Several chromosomally expressed AceE variants were constructed in Escherichia coli ΔldhA ΔpoxB ΔppsA and compared using glucose as the sole carbon source. These variants were examined in shake flask cultures for growth rate, pyruvate accumulation, and acetoin production via heterologous expression of the budA and budB genes from Enterobacter cloacae ssp. dissolvens. The best acetoin‐producing strains were subsequently studied in controlled batch culture at the one‐liter scale. PDH variant strains attained up to four‐fold greater acetoin than the strain expressing the wild‐type PDH. In a repeated batch process, the H106V PDH variant strain attained over 43 g/L of pyruvate‐derived products, acetoin (38.5 g/L) and 2R,3R‐butanediol (5.0 g/L), corresponding to an effective concentration of 59 g/L considering the dilution. The acetoin yield from glucose was 0.29 g/g with a volumetric productivity of 0.9 g/L·h (0.34 g/g and 1.0 g/L·h total products). The results demonstrate a new tool in pathway engineering, the modification of a key metabolic enzyme to improve the formation of a product via a kinetically slow, introduced pathway. Direct modification of the pathway enzyme offers an alternative to promoter engineering in cases where the promoter is involved in a complex regulatory network.