α-Crystallin is a multimeric protein complex which is constitutively expressed at high levels in the vertebrate eye lens, where it serves a structural role, and at low levels in several ...non-lenticular tissues. Like other members of the small heat shock protein family, α-crystallin has a chaperone-like activity in suppressing nonspecific aggregation of denaturing proteins in vitro. Apart from the major αA- and αB-subunits, α-crystallin of rodents contains an additional minor subunit resulting from alternative splicing, αAins-crystallin. This polypeptide is identical to normal αA-crystallin except for an insert peptide of 23 residues. To explore the structural and functional consequences of this insertion, we have expressed rat αA- and αAins-crystallin in Escherichia coli. The multimeric particles formed by αAins are larger and more disperse than those of αA, but they are native-like and display a similar thermostability and morphology, as revealed by gel permeation chromatography, tryptophan fluorescence measurements, and electron microscopy. However, as compared with αA, the αAins-particles display a diminished chaperone-like activity in the protection of heat-induced aggregation of βlow-crystallin. Our experiments indicate that αAins-multimers have a 3-4-fold reduced substrate binding capacity, which might be correlated to their increased particle size and to a shielding of binding sites by the insert peptides. The structure-function relationship of the natural mutant αAins-crystallin may shed light on the mechanism of chaperone-like activity displayed by all small heat shock proteins.
α‐Crystallins are members of the family of small heat‐shock proteins. The conformation and mode of action of these ‘junior chaperones’ are unknown. To investigate the structure and chaperone‐like ...activity, four mutants of bovine αA‐crystallin were generated by site‐directed mutagenesis. In comparison with wild‐type αA‐crystallin, the D69S mutant, in which a highly conserved charged residue has been replaced, forms larger multimers and displays a threefold reduced heat‐protection capacity. The conformation and thermal stability of this mutant are not noticeably affected. Three other mutations, replacing hydrophobic by uncharged hydrophilic residues, were aimed at disturbing hydrophobic intersubunit interactions. None of these mutations resulted in major structural perturbations and only minor differences in heat‐protective capacity were observed. Although it is assumed that small heat‐shock proteins interact with denaturing proteins via their hydrophobic surfaces, this study clearly shows that charged residues in α‐crystallin can also influence the efficiency of substrate binding.
The amine‐donor substrate specificity of tissue‐type transglutaminase has been studied in a series of recombinant αA‐crystallin mutants. These mutant proteins have been provided with a potential ...substrate lysine residue, flanked by different amino acid residues, in the C‐terminal extended arm of αA‐crystallin. A biotinylated amine‐acceptor hexapeptide was used as a probe for labelling the amine‐donor sites. Wild‐type bovine αA‐crystallin does not function as an amine‐donor substrate for tissue‐type transglutaminase. Yet, upon introduction of a lysine residue at the C‐terminal or penultimate position, all mutant αA‐crystallins act as amine‐donor substrates, although to different extents. This shows that accessibility is the primary requirement for a lysine residue to function as an amine‐donor substrate for transglutaminase and that the enzyme has a broad tolerance towards the neighbouring residues. However, the nature of the flanking amino acid residues does clearly affect the reactivity of the substrate lysine residue. Notably, we found that a proline or glycine residue in front of the substrate lysine has a strong adverse effect on the substrate reactivity as compared to a preceding leucine, serine, alanine or arginine residue.
The taxonomy and systematic relationships among species of Solanum section Petota are complicated and the section seems overclassified. Many of the presumed (sub)species from South America are very ...similar and they are able to exchange genetic material. We applied a population genetic approach to evaluate support for subgroups within this material, using AFLP data. Our approach is based on the following assumptions: (i) accessions that may exchange genetic material can be analyzed as if they are part of one gene pool, and (ii) genetic differentiation among species is expected to be higher than within species.
A dataset of 566 South-American accessions (encompassing 89 species and subspecies) was analyzed in two steps. First, with the program STRUCTURE 2.2 in an 'unsupervised' procedure, individual accessions were assigned to inferred clusters based on genetic similarity. The results showed that the South American members of section Petota could be arranged in 16 clusters of various size and composition. Next, the accessions within the clusters were grouped by maximizing the partitioning of genetic diversity among subgroups (i.e., maximizing Fst values) for all available individuals of the accessions (2767 genotypes). This two-step approach produced an optimal partitioning into 44 groups.Some of the species clustered as genetically distinct groups, either on their own, or combined with one or more other species. However, accessions of other species were distributed over more than one cluster, and did not form genetically distinct units.
We could not find any support for 43 species (almost half of our dataset). For 28 species some level of support could be found varying from good to weak. For 18 species no conclusions could be drawn as the number of accessions included in our dataset was too low. These molecular data should be combined with data from morphological surveys, with geographical distribution data, and with information from crossing experiments to identify natural units at the species level. However, the data do indicate which taxa or combinations of taxa are clearly supported by a distinct set of molecular marker data, leaving other taxa unsupported. Therefore, the approach taken provides a general method to evaluate the taxonomic system in any species complex for which molecular data are available.
Mitochondrial or oxidative phosphorylation diseases are relatively frequent, multisystem disorders; in about 15% of cases they are caused by maternally inherited mitochondrial DNA (mtDNA) mutations. ...Because of the possible severity of the phenotype, the lack of effective treatment, and the high recurrence risk for offspring of carrier females, couples wish to prevent the transmission of these mtDNA disorders to their offspring. Prenatal diagnosis is problematic for several reasons, and concern the often poor correlation between mutation percentages and disease severity and the uncertainties about the representativeness of a fetal sample. A new option for preventing transmission of mtDNA disorders is preimplantation genetic diagnosis (PGD), which circumvents these problems by transferring an embryo below the threshold of clinical expression.
We present the data on nine PGD cycles in four female carriers of mitochondrial diseases: three mitochondrial encephalopathy, lactic acidosis and stroke-like episodes (MELAS) (m.3243A>G), and one Leigh (m.8993T>G). Our threshold for transfer after PGD is 15% for the m.3243A>G mutation and 30% for the m.8993T>G mutation.
All four female carriers produced embryos eligible for transfer. The m.8993T>G mutation in oocytes/embryos showed more skewing than the m.3243A>G. In about 80% of the embryos the mutation load in the individual blastomeres was fairly constant (interblastomere differences <10%). However, in around 11% (in embryos with the m.3243A>G mutation only), the mutation load differed substantially (>15%) between blastomeres of a single embryo, mostly as a result of one outlier. The m.8993T>G carrier became pregnant and gave birth to a healthy son.
PGD provides carriers of mtDNA mutations the opportunity to conceive healthy offspring.
Abstract
Objective
RA is associated with higher risk of cardiovascular (CV) disease. Ongoing systemic inflammation is presumed to accelerate atherosclerosis by increasing inflammation in the arterial ...wall. However, evidence supporting this hypothesis is limited. We aimed to investigate arterial wall inflammation in RA vs OA, and its association with markers of inflammation and CV risk factors.
Methods
18-fluorodeoxyglucose PET combined with CT (18F-FDG-PET/CT) was performed in RA (n = 61) and OA (n = 28) to investigate inflammatory activity in the wall of large arteries. Secondary analyses were performed in patients with early untreated RA (n = 30), and established RA, active under DMARD treatment (n = 31) vs OA.
Results
Patients with RA had significantly higher 18F-FDG uptake in the wall of the carotid arteries (beta 0.27, 95%CI 0.11—0.44, P <0.01) and the aorta (beta 0.47, 95%CI 0.17—0.76, P <0.01) when compared with OA, which persisted after adjustment for traditional CV risk factors. Patients with early RA had the highest 18F-FDG uptake, followed by patients with established RA and OA respectively. Higher ESR and DAS of 28 joints values were associated with higher 18F-FDG uptake in all arterial segments.
Conclusion
Patients with RA have increased 18F-FDG uptake in the arterial wall compared with patients with OA, as a possible marker of early atherosclerosis. Furthermore, a higher level of clinical disease activity and circulating inflammatory markers was associated with higher arterial 18F-FDG uptake, which may support a role of arterial wall inflammation in the pathogenesis of vascular complications in patients with RA.
To evaluate the feasibility and anatomical success of endovenous laser ablation (EVLA) of incompetent perforating veins (IPV).
All 135 consecutive patients with IPV treated with ELVA ...(intention-to-treat) from January 2008 to December 2013 were included. Up to the end of 2011, an 810-nm laserset (14 W) was used, and afterwards, a 1470-nm laserset (6 W) was introduced. Duplex ultrasound was performed at 6 weeks' follow-up to assess anatomical success.
Overall anatomical success at 6 weeks' follow-up was 56%. Anatomical success was 63% after treatment with 810 nm and 45% with 1470 nm (p = 0.035). This difference in the success rate seems associated with the significantly higher amount of energy delivered in the 810 nm cohort (560 J) versus 1470 nm (186 J). Regardless of the type of laser, anatomical success was significantly higher after treatment with more than 400 J (66%) compared with 0-200 J (40%, p = 0.009) and 200-400 J (43%, p = 0.029). Complications were limited to two cases of transient paresthesia.
EVLA of IPVs is safe and feasible. The amount of energy is highly important in achieving anatomical success.
Endothelial dysfunction and low-grade inflammation are associated with cardiovascular disease. Arterial stiffening plays an important role in cardiovascular disease and, thus, may be a mechanism ...through which endothelial dysfunction and/or low-grade inflammation lead to cardiovascular disease. We investigated the associations between, on the one hand, biomarkers of endothelial dysfunction (soluble endothelial selectin, thrombomodulin, and both vascular and intercellular adhesion molecules 1 and von Willebrand factor) and of low-grade inflammation (C-reactive protein, serum amyloid A, interleukin 6, interleukin 8, tumor necrosis factor-α and, soluble intercellular adhesion molecule 1) and, on the other hand, arterial stiffness over a 6-year period, in 293 healthy adults (155 women). Biomarkers were combined into mean z scores. Carotid, femoral, and brachial arterial stiffness and carotid-femoral pulse wave velocity were determined by ultrasonography. Measurements were obtained when individuals were 36 and 42 years of age. Associations were analyzed with generalized estimating equation and adjusted for sex, height, and mean arterial pressure. The endothelial dysfunction z score was inversely associated with femoral distensibility (β: -0.51 95% CI: -0.95 to -0.06) and compliance coefficients (β: -0.041 95% CI: -0.076 to -0.006) but not with carotid or brachial stiffness or carotid-femoral pulse wave velocity. The low-grade inflammation z score was inversely associated with femoral distensibility (β: -0.51 95% CI: -0.95 to -0.07) and compliance coefficients (β: -0.050 95% CI: -0.084 to -0.016) and with carotid distensibility coefficient (β: -0.910 95% CI: -1.810 to -0.008) but not with brachial stiffness or carotid-femoral pulse wave velocity. Biomarkers of endothelial dysfunction and low-grade inflammation are associated with greater arterial stiffness. This provides evidence that arterial stiffening may be a mechanism through which endothelial dysfunction and low-grade inflammation lead to cardiovascular disease.
Abstract
We report on the first PGD performed for the m.14487 T>C mitochondrial DNA (mtDNA) mutation in the MT-ND6 gene, associated with Leigh syndrome. The female carrier gave birth to a healthy ...baby boy at age 42. This case adds to the successes of PGD for mtDNA mutations.
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