Genomic and experimental evidence for multiple metabolic functions in the RidA/YjgF/YER057c/UK114 (Rid) protein family

Background

It is now recognized that enzymatic or chemical side-reactions can convert normal metabolites to useless or toxic ones and that a suite of enzymes exists to mitigate such metabolite damage. Examples are the reactive imine/enamine intermediates produced by threonine dehydratase, which damage the pyridoxal 5''-phosphate cofactor of various enzymes causing inactivation. This damage is pre-empted by RidA proteins, which hydrolyze the imines before they do harm. RidA proteins belong to the YjgF/YER057c/UK114 family (here renamed the Rid family). Most other members of this diverse and ubiquitous family lack defined functions.

Results

Phylogenetic analysis divided the Rid family into a widely distributed, apparently archetypal RidA subfamily and seven other subfamilies (Rid1 to Rid7) that are largely confined to bacteria and often co-occur in the same organism with RidA and each other. The Rid1 to Rid3 subfamilies, but not the Rid4 to Rid7 subfamilies, have a conserved arginine residue that, in RidA proteins, is essential for imine-hydrolyzing activity. Analysis of the chromosomal context of bacterial RidA genes revealed clustering with genes for threonine dehydratase and other pyridoxal 5''-phosphate-dependent enzymes, which fits with the known RidA imine hydrolase activity. Clustering was also evident between Rid family genes and genes specifying FAD-dependent amine oxidases or enzymes of carbamoyl phosphate metabolism. Biochemical assays showed that Salmonella enterica RidA and Rid2, but not Rid7, can hydrolyze imines generated by amino acid oxidase. Genetic tests indicated that carbamoyl phosphate overproduction is toxic to S. enterica cells lacking RidA, and metabolomic profiling of Rid knockout strains showed ten-fold accumulation of the carbamoyl phosphate-related metabolite dihydroorotate.

Conclusions

Like the archetypal RidA subfamily, the Rid2, and probably the Rid1 and Rid3 subfamilies, have imine-hydrolyzing activity and can pre-empt damage from imines formed by amine oxidases as well as by pyridoxal 5''-phosphate enzymes. The RidA subfamily has an additional damage pre-emption role in carbamoyl phosphate metabolism that has yet to be biochemically defined. Finally, the Rid4 to Rid7 subfamilies appear not to hydrolyze imines and thus remain mysterious.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1584-3) contains supplementary material, which is available to authorized users.     

Optimizing Human Synovial Fluid Preparation for Two-Dimensional Gel Electrophoresis

Background

Prenatal screening for Down Syndrome (DS) would benefit from an increased number of biomarkers to improve sensitivity and specificity. Improving sensitivity and specificity would decrease the need for potentially risky invasive diagnostic procedures.

Results

We have performed an in depth two-dimensional difference gel electrophoresis (2D DIGE) study to identify potential biomarkers. We have used maternal plasma samples obtained from first and second trimesters from mothers carrying DS affected fetuses compared with mothers carrying normal fetuses. Plasma samples were albumin/IgG depleted and expanded pH ranges of pH 4.5 - 5.5, pH 5.3 - 6.5 and pH 6 - 9 were used for two-dimensional gel electrophoresis (2DE). We found no differentially expressed proteins in the first trimester between the two groups. Significant up-regulation of ceruloplasmin, inter-alpha-trypsin inhibitor heavy chain H4, complement proteins C1s subcomponent, C4-A, C5, and C9 and kininogen 1 were detected in the second trimester in maternal plasma samples where a DS affected fetus was being carried. However, ceruloplasmin could not be confirmed as being consistently up-regulated in DS affected pregnancies by Western blotting.

Conclusions

Despite the in depth 2DE approach used in this study the results underline the deficiencies of gel-based proteomics for detection of plasma biomarkers. Gel-free approaches may be more productive to increase the number of plasma biomarkers for DS for non-invasive prenatal screening and diagnosis.     

Arsenic, cadmium and neuron specific enolase (ENO2, γ-enolase) expression in breast cancer

Background

Neuron specific enolase (ENO2, γ-enolase) has been used as a biomarker to help identify neuroendocrine differentiation in breast cancer. The goal of the present study was to determine if ENO2 expression in the breast epithelial cell is influenced by the environmental pollutants, arsenite and cadmium. Acute and chronic exposure of MCF-10A cells to As⁺³ and Cd⁺² sufficient to allow colony formation in soft agar, was used to determine if ENO2 expression was altered by these pollutants.

Results

It was shown that both As⁺³ and Cd⁺² exposure caused significant increases in ENO2 expression under conditions of both acute and chronic exposure. In contrast, ENO1, the major glycolytic enolase in non-muscle and neuronal cells, was largely unaffected by exposure to either As⁺³ or Cd⁺². Localization studies showed that ENO2 in the MCF-10A cells transformed by As⁺³ or Cd⁺² had both a cytoplasmic and nuclear localization. In contrast, ENO1 was localized to the cytoplasm. ENO2 localized to the cytoplasm was found to co-localized with ENO1.

Conclusion

The results are the first to show that ENO2 expression in breast epithelial cells is induced by acute and chronic exposure to As⁺³ or Cd⁺². The findings also suggest a possible link between As⁺³ and Cd⁺² exposure and neuroendocrine differentiation in tumors. Overall, the results suggest that ENO2 might be developed as a biomarker indicating acute and/or chronic environmental exposure of the breast epithelial cell to As⁺³ and Cd⁺².     

Probing the limits of aptamer affinity with a microfluidic SELEX platform

Nucleic acid-based aptamers offer many potential advantages relative to antibodies and other protein-based affinity reagents, including facile chemical synthesis, reversible folding, improved thermal stability and lower cost. However, their selection requires significant time and resources and selections often fail to yield molecules with affinities sufficient for molecular diagnostics or therapeutics. Toward a selection technique that can efficiently and reproducibly generate high performance aptamers, we have developed a microfluidic selection process (M-SELEX) that can be used to obtain high affinity aptamers against diverse protein targets. Here, we isolated DNA aptamers against three protein targets with different isoelectric points (pI) using a common protocol. After only three rounds of selection, we discovered novel aptamer sequences that bind to platelet derived growth factor B (PDGF-BB; pI = 9.3) and thrombin (pI = 8.3) with respective dissociation constants (K_d) of 0.028 nM and 0.33 nM, which are both superior to previously reported aptamers against these targets. In parallel, we discovered a new aptamer that binds to apolipoprotein E3 (ApoE; pI = 5.3) with a K_d of 3.1 nM. Furthermore, we observe that the net protein charge may exert influence on the affinity of the selected aptamers. To further explore this relationship, we performed selections against PDGF-BB under different pH conditions using the same selection protocol, and report an inverse correlation between protein charge and aptamer K_d.     

Protozoan parasite Toxoplasma gondii manipulates mate choice in rats by enhancing attractiveness of males

Females in various species typically avoid males infected with parasites, while parasite-free males advertise their status through conspicuous phenotypic traits. This process selects for heritable resistance and reduces direct exposure of the female to parasites. Coevolving parasites are likely to attempt to circumvent this obstacle. In this paper, we demonstrate a case of parasitic manipulation of host mate choice. We report that Toxoplasma gondii, a sexually transmitted infection of brown rats, enhances sexual attractiveness of infected males. Thus under some evolutionary niches, parasites can indeed manipulate host sexual signaling to their own advantage.     

Changes in ultraweak photon emission and heart rate variability of epinephrine-injected rats

Ultraweak photons which are spontaneously emitted from a living body may be applicable as a non-invasive tool to characterize the physiological state of the living body. We investigated changes in the intensity of ultraweak photon emission, body temperature and the cardiovascular autonomic activity induced by epinephrine injection to rats. A high dose of epinephrine can make changes to the cardiovascular autonomic activity or body temperature. Photon emission of the dorsal part, rectal temperature and heart rate variability (HRV) were measured from eight Sprague-Dawley rats. The intensities of photon emissions for saline injections, which were used as a control, decreased from 13042+/-71 counts/min at the start of measurements to 8709+/-915 counts/min at 1 h after the injections. In the case with epinephrine injections, the intensity of photon emission reduced slowly from 13361+/-354 counts/min to 11040+/-433 counts/min. Rectal temperature increased in both saline- and epinephrine-injected rats, but one hour after the injections the temperature in the epinephrine case was slightly higher than that in the saline case. The standard deviation of the QRS wave complex interval (RR interval) increased from 1 to 4 (p<0.05) and the spectral ratio of the low frequency component to the high frequency component in the HRV data LF (0.19 approximately 0.74 Hz) / HF (0.78 approximately 2.50 Hz) decreased from 0.81 to 0.26 (p<0.05) in the case of epinephrine injection while no change was found in the case of saline injection. Thus, ultraweak photon emission was closely related to the cardiovascular autonomic activity.     

Relationship of number of remaining teeth to health-related quality of life in community-dwelling elderly

Objective: The aim of this study was to evaluate the relationship between number of remaining teeth and health‐related quality of life in community‐dwelling elderly. Subjects: A total of 207 participants who were community‐dwelling, 85 years of age. Data were from a population‐based study of age‐related general and oral health in Fukuoka Prefecture, Japan. Measurements: The Japanese version of the Short Form 36 Health Survey (SF‐36). Results: The mental component score for the participants, from the SF‐36, was higher than the Japanese national norm for those aged ≥70 years. There were no significant differences in the mean of any scores on the SF‐36 by having spouse, living with family, or education level. The mean of the SF‐36 scores of physical functioning (PF) and of the physical component scores were significantly higher in the 85‐year‐old participants with ≥20 teeth than in those with ≤19 teeth (p < 0.05 and p < 0.01 respectively). In addition, a significant difference (p < 0.05) was observed between the mean of participants with ≥20 teeth and those with ≤19 teeth after adjustment for region where the participant lived, activities of daily living (ADL), and sex. The PF (p < 0.001), role‐physical (p < 0.005), bodily pain (p < 0.001), vitality (p < 0.001), social functioning (p < 0.05), and physical component (p < 0.001) scores were significantly higher in participants with a good activities of daily living (ADL) assessment. However, ADL was not associated with the number of teeth. Conclusions: The findings of the present study indicated that 85‐year‐old participants with ≥20 teeth had better subjective physical health than those with ≤19 teeth.     

A population-based LD map of the human chromosome 6p

The recent publication of the complete sequence of human chromosome 6 provides a platform from which to investigate genomic sequence variation. We report here a detailed linkage disequilibrium (LD) pattern map across the entire human chromosome 6p by using a set of 1152 single nucleotide polymorphisms (SNPs) in a population of 198 Singaporean Chinese, with 326 SNPs focused in the major histocompatibility complex (MHC) region. Our analysis shows some unexpectedly high segments of strong LD in a 10-Mb region that includes the extremely polymorphic and gene-rich MHC loci and many non-MHC genes. These include the telomeric peri-MHC region that harbors olfactory receptors, histones and zinc finger clusters, and the centromeric peri-MHC region that contains several unknown open reading frames. The data also help refine a human–mouse synteny break in the region between 28.6 and 29.4 Mb. The population-based LD map presented here will provide an essential resource for understanding the genomic sequence variation of chromosome 6p and LD mapping of disease genes of complex genetic traits. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users. H. Yu and J.-M. Chia should be regarded as joint first authors.