Sensory Ataxic Neuropathy in Golden Retriever Dogs Is Caused by a Deletion in the Mitochondrial tRNATyr Gene

Sensory ataxic neuropathy (SAN) is a recently identified neurological disorder in golden retrievers. Pedigree analysis revealed that all affected dogs belong to one maternal lineage, and a statistical analysis showed that the disorder has a mitochondrial origin. A one base pair deletion in the mitochondrial tRNA^Tyr gene was identified at position 5304 in affected dogs after re-sequencing the complete mitochondrial genome of seven individuals. The deletion was not found among dogs representing 18 different breeds or in six wolves, ruling out this as a common polymorphism. The mutation could be traced back to a common ancestor of all affected dogs that lived in the 1970s. We used a quantitative oligonucleotide ligation assay to establish the degree of heteroplasmy in blood and tissue samples from affected dogs and controls. Affected dogs and their first to fourth degree relatives had 0–11% wild-type (wt) sequence, while more distant relatives ranged between 5% and 60% wt sequence and all unrelated golden retrievers had 100% wt sequence. Northern blot analysis showed that tRNA^Tyr had a 10-fold lower steady-state level in affected dogs compared with controls. Four out of five affected dogs showed decreases in mitochondrial ATP production rates and respiratory chain enzyme activities together with morphological alterations in muscle tissue, resembling the changes reported in human mitochondrial pathology. Altogether, these results provide conclusive evidence that the deletion in the mitochondrial tRNA^Tyr gene is the causative mutation for SAN.     

POSTMan (POST‐translational modification analysis), a software application for PTM discovery

Post‐translationally modified peptides present in low concentrations are often not selected for CID, resulting in no sequence information for these peptides. We have developed a software POSTMan (POST‐translational Modification analysis) allowing post‐translationally modified peptides to be targeted for fragmentation. The software aligns LC‐MS runs (MS₁ data) between individual runs or within a single run and isolates pairs of peptides which differ by a user defined mass difference (post‐translationally modified peptides). The method was validated for acetylated peptides and allowed an assessment of even the basal protein phosphorylation of phenylalanine hydroxylase (PHA) in intact cells.     

Oxidation of Lanthionines Renders the Lantibiotic Nisin Inactive

The peptide antibiotic nisin A belongs to the group of antibiotics called lantibiotics. They are classified as lantibiotics because they contain the structural group lanthionine. Lanthionines are composed of a single sulfur atom that is linked to the β-carbons of two alanine moieties. These sulfur atoms are vulnerable to environmental oxidation. A mild oxidation reaction was performed on nisin A to determine the relative effects it would have on bioactivity. High-mass-accuracy Fourier transform ion cyclotron resonance mass spectrometry data revealed the addition of seven, eight, and nine oxygens. These additions correspond to the five lanthionines, two methionines, and two histidines that would be susceptible to oxidation. Subsequent bioassays revealed that the oxidized form of nisin A had a complete loss of bactericidal activity. In a competition study, the oxidized nisin did not appear to have an antagonistic affect on the bioactivity of nisin A, since the addition of an equal molar concentration of the oxidized variant did not have an influence on the bactericidal activity of the native antibiotic. Electron microscopy data revealed that the oxidized forms were still capable of assembling into large circular complexes, demonstrating that oxidation does not disrupt the lateral assembly mechanism of the antibiotic. Affinity thin-layer chromatography and fluorescence microscopy experiments suggested that the loss of activity is due to the inability of the oxidized form of nisin to bind to the cell wall precursor lipid II. Given the loss of bioactivity following oxidation, oxidation should be an important factor to consider in future production, purification, pharmacokinetic, and pharmacodynamic studies.Lantibiotics are ribosomally synthesized peptide bacteriocins that undergo extensive posttranslational modifications to yield unusual amino acids, like lanthionine, methyllanthionine, 2,3-didehydroalanine, 2,3-didehydrobutyrine, and S-[aminovinyl] cysteine (8). The name lantibiotic is derived from the presence of the posttranslationally modified lanthionine residues. Nisin A (3,351.5 Da), produced by Lactococcus lactis, belongs to this class of antibiotics and is further subclassified as a type A(I) lantibiotic. Type A(I) lantibiotics are cationic and have a rigid ring conformation separated by areas of flexibility. Another well-studied lantibiotic, gallidermin, also belongs to this class of lantibiotics and has significant homology to nisin A in the first two lanthionine rings, A and B (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Schematic of the covalent structures of nisin A and gallidermin. The N-terminal rings A and B are believed to be responsible for binding to lipid II.The antibiotics in this class have drawn considerable attention for their bactericidal potential as preservatives and for their potential for treating Staphylococcus and Streptococcus infections. Nisin A has been used for over 40 years in Europe as a preservative in the food industry and was approved for use in the United States by the FDA in 1988. Its uses include controlling the growth of various bacteria in pasteurized cheese and liquid egg ingredients, as well as preserving salad dressings (12), canned foods (10, 32), and, most recently, ground beef (24). Other lantibiotics, including gallidermin and epidermin, have been shown to be useful as treatments for acne and in the maintenance of oral health (19, 21). Recent literature shows that both nisin A and gallidermin can be used to treat and/or prevent mastitis in bovines (3, 25a), and they are currently marketed as wipes.Lantibiotics have multiple modes of bactericidal activity (2, 16). In the case of nisin A, the sensitivity of the host bacterium has been shown to be dependent on the charge states of its cell wall and membrane (1, 6, 25). More importantly, the bactericidal activity is attributed to lipid II abduction (4, 5, 7). A novel mechanism of antimicrobial activity for nisin A has been described, in which it binds to lipid II and sequesters it into large complexes. These complexes aid in the abduction of lipid II from the growth zones of bacteria, where lipid II is required for new cell wall formation (16). A novel lipid II binding motif for nisin A has been characterized by nuclear magnetic resonance (NMR) (18) in which the N-terminal portion of nisin A, lanthionine rings A and B, interacts with the pyrophosphate, the peptidoglycan MurNAc, and the first isoprene of lipid II.An oxidized form of nisin A was characterized using bactericidal assays, high-mass-accuracy Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS), chromatography methods, and electron and fluorescence microscopy techniques. The objectives of this study were to determine changes in the biophysical properties of oxidized nisin A as they relate to its bactericidal activity, its ability to interact with bacterial membranes, its capacity for lateral assembly, and its ability to bind to lipid II.     

Analysis of the rat Iddm14 diabetes susceptibility locus in multiple rat strains: identification of a susceptibility haplotype in the Tcrb-V locus

Iddm14 (formerly Iddm4) is a non-MHC-linked genetic locus associated with autoimmune diabetes. Its effects have been well-documented in BB-derived rats in which diabetes is either induced by immunologic perturbation or occurs spontaneously. The role of Iddm14 in non-BB rat strains is unknown. Our goal was to extend the analysis of Iddm14 in new diabetes-susceptible strains and to identify candidate genes in the rat Iddm14 diabetes susceptibility locus that are common to these multiple diabetic strains. To determine if Iddm14 is important in strains other than BB, we first genotyped a (LEW.1WR1 × WF)F2 cohort in which diabetes was induced by perturbation with polyinosinic:polycytidylic acid. We found that Iddm14 is a major determinant of diabetes susceptibility in LEW.1WR1 rats. We then used nucleotide sequencing to establish a strain distribution pattern of polymorphisms (insertions, deletions, and single nucleotide polymorphisms [SNPs]) that predicts susceptibility to diabetes in a panel of inbred and congenic rats. Using the positional information from the congenic strains and the new linkage data, we identified a susceptibility haplotype in the T-cell receptor Vβ chain (Tcrb-V) locus. This haplotype includes Tcrb-V13, which is identical in five susceptible strains but different in resistant WF and F344 rats. We conclude that Iddm14 is a powerful determinant of both spontaneous and induced autoimmune diabetes in multiple rat strains, and that Tcrb-V13 SNPs constitute a haplotype of gene elements that may be critical for autoimmune diabetes in rats.     

Fibrocytes are associated with vascular and parenchymal remodelling in patients with obliterative bronchiolitis

Background

The aim of the present study was to explore the occurrence of fibrocytes in tissue and to investigate whether the appearance of fibrocytes may be linked to structural changes of the parenchyme and vasculature in the lungs of patients with obliterative bronchiolitis (OB) following lung or bone marrow transplantation.

Methods

Identification of parenchyme, vasculature, and fibrocytes was done by histological methods in lung tissue from bone marrow or lung-transplanted patients with obliterative bronchiolitis, and from controls.

Results

The transplanted patients had significantly higher amounts of tissue in the alveolar parenchyme (46.5 ± 17.6%) than the controls (21.7 ± 7.6%) (p < 0.05). The patients also had significantly increased numbers of fibrocytes identified by CXCR4/prolyl4-hydroxylase, CD45R0/prolyl4-hydroxylase, and CD34/prolyl4-hydroxylase compared to the controls (p < 0.01). There was a correlation between the number of fibrocytes and the area of alveolar parenchyma; CXCR4/prolyl 4-hydroxylase (p < 0.01), CD45R0/prolyl 4-hydroxylase (p < 0.05) and CD34/prolyl 4-hydroxylase (p < 0.05). In the pulmonary vessels, there was an increase in the endothelial layer in patients (0.31 ± 0.13%) relative to the controls (0.037 ± 0.02%) (p < 0.01). There was a significant correlation between the number of fibrocytes and the total area of the endothelial layer CXCR4/prolyl 4-hydroxylase (p < 0.001), CD45R0/prolyl 4-hydroxylase (p < 0.001) and CD34/prolyl 4-hydroxylase (p < 0.01). The percent areas of the lumen of the vessels were significant (p < 0.001) enlarged in the patient with OB compared to the controls. There was also a correlation between total area of the lumen and number of fibrocytes, CXCR4/prolyl 4-hydroxylase (p < 0.01), CD45R0/prolyl 4-hydroxylase (p < 0.001) and CD34/prolyl 4-hydroxylase (p < 0.01).

Conclusion

Our results indicate that fibrocytes are associated with pathological remodelling processes in patients with OB and that tissue fibrocytes might be a useful biomarker in these processes.     

Genetic Architecture of Tameness in a Rat Model of Animal Domestication

A common feature of domestic animals is tameness—i.e., they tolerate and are unafraid of human presence and handling. To gain insight into the genetic basis of tameness and aggression, we studied an intercross between two lines of rats (Rattus norvegicus) selected over >60 generations for increased tameness and increased aggression against humans, respectively. We measured 45 traits, including tameness and aggression, anxiety-related traits, organ weights, and levels of serum components in >700 rats from an intercross population. Using 201 genetic markers, we identified two significant quantitative trait loci (QTL) for tameness. These loci overlap with QTL for adrenal gland weight and for anxiety-related traits and are part of a five-locus epistatic network influencing tameness. An additional QTL influences the occurrence of white coat spots, but shows no significant effect on tameness. The loci described here are important starting points for finding the genes that cause tameness in these rats and potentially in domestic animals in general.ANIMAL domestication marked a turning point in human prehistory (Diamond 2002), and domestic animals have been the subject of research for many years (Darwin 1868). Recently, genetic studies have shed light on when, where, and how often a range of animal species were domesticated (Troy et al. 2001; Vila et al. 2001; Savolainen et al. 2002; Larson et al. 2005; Driscoll et al. 2007; Eriksson et al. 2008; Naderi et al. 2008). With the exception of coat color (e.g., Pielberg et al. 2008) and skin pigmentation (Eriksson et al. 2008), little is known about what occurred genetically during animal domestication. At what genes were allelic variants selected for by would-be practitioners of animal husbandry? Although domestic animals differ from each other in many ways, they all share the trait of tameness—i.e., they tolerate and sometimes even seek human presence and handling. Almost nothing is currently known about the genetic basis of tameness.In a series of studies initiated by D. K. Belyaev, researchers at the Institute for Cytology and Genetics in Novosibirsk (Russia) have subjected several mammalian species to a process of experimental domestication (Trut 1999). These studies, some of them ongoing for several decades, involve selection for tame and aggressive behavior in lines of animals derived from wild populations. They include a fox population that has been “domesticated” to such an extent that the tame foxes are now similar to dogs in some respects (Hare et al. 2005). They also include a population of wild-caught rats (Rattus norvegicus) that was selected for either reduced or enhanced aggression toward humans over >60 generations (Belyaev and Borodin 1982). To select the animals, their response to an approaching human hand was observed, and the rats showing the least and the most aggressive behavior were allowed to mate within the two lines, respectively. The initial response to selection was rapid and then slowed, so that little change in behavior from generation to generation has been observed in the last 10–15 generations, although the selection regime has been continued to the present. Today, the “tame” rats are completely unafraid of humans, they tolerate handling and being picked up, and they sometimes approach a human in a nonaggressive manner. By contrast, the “aggressive” rats ferociously attack or flee from an approaching human hand.To study the genetic basis of tameness we have established populations of both rat lines in Leipzig. In their new environment, the rats maintained their behavioral differences in response to humans, and these differences were not influenced by postnatal maternal factors (Albert et al. 2008). In addition, the rat lines differ in a number of other behavioral, anatomical, and physiological traits, raising the question whether these traits are influenced by the same loci as tameness and aggression toward humans.Many domestic animals display conspicuous coat color variations not found in their wild relatives. Prominent examples include the white color variants in dogs, pigs, cows, horses, and chickens. In laboratory rats, it has been proposed that “coat color genes” may account for many of the differences associated with domestication (Keeler and King 1942). It is thus interesting that individuals with white spots appeared in both the tame foxes (Trut 1999) and the tame rats (Trut et al. 2000) at higher frequency than in the corresponding aggressive lines, although they were absent or rare in the founding fox and rat populations, and although they were not selected for. The rat populations studied here provide an excellent opportunity to examine whether tameness is influenced by the same loci as white coat spotting.In this study, we crossed the two rat lines and bred >700 intercross animals. A broad set of behavioral, anatomical, and physiological traits was measured, and a genomewide set of genetic markers was used to identify genomic regions (quantitative trait loci, QTL) that influence tameness as well as other traits that differ between the lines, including white spots.     

Mimicking the electron donor side of Photosystem II in artificial photosynthesis

This review focuses on our recent efforts in synthetic ruthenium–tyrosine–manganese chemistry mimicking the donor side reactions of Photosystem II. Tyrosine and tryptophan residues were linked to ruthenium photosensitizers, which resulted in model complexes for proton-coupled electron transfer from amino acids. A new mechanistic model was proposed and used to design complexes in which the mechanism could be switched between concerted and step-wise proton-coupled electron transfer. Moreover, a manganese dimer linked to a ruthenium complex could be oxidized in three successive steps, from Mn₂^II,II to Mn₂^III,IV by the photo-oxidized ruthenium sensitizer. This was possible thanks to a charge compensating ligand exchange in the manganese complex. Detailed studies of the ligand exchange suggested that at high water concentrations, each oxidation step is coupled to a proton-release of water-derived ligands, analogous to the oxidation steps of the manganese cluster of Photosystem II.     

Chimeric antibody-binding Vitreoscilla hemoglobin (VHb) mediates redox-catalysis reaction: new insight into the functional role of VHb

Experimentation was initiated to explore insight into the redox-catalysis reaction derived from the heme prosthetic group of chimeric Vitreoscilla hemoglobin (VHb). Two chimeric genes encoding chimeric VHbs harboring one and two consecutive sequences of Fc-binding motif (Z-domain) were successfully constructed and expressed in E. coli strain TG1. The chimeric ZVHb and ZZVHb were purified to a high purity of more than 95% using IgG-Sepharose affinity chromatography. From surface plasmon resonance, binding affinity constants of the chimeric ZVHb and ZZVHb to human IgG were 9.7 x 10(7) and 49.1 x 10(7) per molar, respectively. More importantly, the chimeric VHbs exhibited a peroxidase-like activity determined by activity staining on native PAGE and dot blotting. Effects of pH, salt, buffer system, level of peroxidase substrate and chromogen substrate were determined in order to maximize the catalytic reaction. From our findings, the chimeric VHbs displayed their maximum peroxidase-like activity at the neutral pH (approximately 7.0) in the presence of high concentration (20-40 mM) of hydrogen peroxide. Under such conditions, the detection limit derived from the calibration curve was at 250 ng for the chimeric VHbs, which was approximately 5-fold higher than that of the horseradish peroxidase. These findings reveal the novel functional role of Vitreoscilla hemoglobin indicating a high trend of feasibility for further biotechnological and medical applications.