Nuclear DNA content variation among perennial taxa of the genus Cyanus (Asteraceae) in Central Europe and adjacent areas

The genome size of 265 plants and the GC content of 126 plants from 63 populations of the Cyanus triumfetti and Cyanus montanus groups, collected across the Carpathians, Pannonia, Bohemian Massif, and Western and Dinaric Alps were determined by PI and DAPI flow cytometry. Variation of the nuclear DNA content among homoploid species, and intraspecific and interpopulation variation were confirmed in simultaneous analyses. The 2C-value at the diploid level (the C. triumfetti group) varied from 2.53 for Cyanus dominii subsp. sokolensis to 3.06?pg for C. triumfetti s.s. (1.21-fold range). At the tetraploid level (the C. montanus group), the 2C-value varied from 5.19 for Cyanus mollis to 5.84?pg for C. montanus (1.13-fold range). High intraspecific and interpopulation variation in the amount of nuclear DNA in the C. triumfetti group correlates with the extensive morphological variation found in this group. Significant between-species differences in genome size indicate that this attribute may be used as a supportive taxonomic marker for both of the groups studied. The GC content varied by 2.93?%, from 39.46?% for “Cyanus axillaris” to 40.61?% for Cyanus adscendens; this character is of no value for taxonomic purposes. Genome size of the studied populations is significantly higher in southern parts of the distribution area and at higher elevations. Plants with smaller genomes tend to occur in dry areas at low altitudes with high diurnal and annual temperature oscillations. The GC content of the populations studied is significantly correlated with longitude, increasing from east to west; and plants with GC-rich genomes are concentrated in the coldest areas with low minimum temperatures.     

Three-dimensional structure of glycolipids in biological membranes

Characterizing the structure-function relationships of glycolipids in lipid membranes is a challenging endeavor. Glycolipid "structure" is rarely, if ever, a unique low-energy conformer, but an ensemble of dynamic states, which vary in their presentation of binding epitopes. The modulation of binding epitopes not only is an internal process but also is influenced by external factors such as glycolipid clustering and fluctuations in and composition of the fluid membrane environment. As with other glyco-conjugates, three-dimensional structural elucidation has relied heavily on nuclear magnetic resonance spectroscopy and computational modeling. Discrete conformational states can be discerned from motion-averaged experimental data by employing independent molecular dynamics simulations. Using model membranes such as micelles, bicelles, and bilayers, we can approximate the effect of their biological environment and quantify cell-surface presentation.     

The TRK-Fused Gene Is Mutated in Hereditary Motor and Sensory Neuropathy with Proximal Dominant Involvement

Hereditary motor and sensory neuropathy with proximal dominant involvement (HMSN-P) is an autosomal-dominant neurodegenerative disorder characterized by widespread fasciculations, proximal-predominant muscle weakness, and atrophy followed by distal sensory involvement. To date, large families affected by HMSN-P have been reported from two different regions in Japan. Linkage and haplotype analyses of two previously reported families and two new families with the use of high-density SNP arrays further defined the minimum candidate region of 3.3 Mb in chromosomal region 3q12. Exome sequencing showed an identical c.854C>T (p.Pro285Leu) mutation in the TRK-fused gene (TFG) in the four families. Detailed haplotype analysis suggested two independent origins of the mutation. Pathological studies of an autopsied patient revealed TFG- and ubiquitin-immunopositive cytoplasmic inclusions in the spinal and cortical motor neurons. Fragmentation of the Golgi apparatus, a frequent finding in amyotrophic lateral sclerosis, was also observed in the motor neurons with inclusion bodies. Moreover, TAR DNA-binding protein 43 kDa (TDP-43)-positive cytoplasmic inclusions were also demonstrated. In cultured cells expressing mutant TFG, cytoplasmic aggregation of TDP-43 was demonstrated. These findings indicate that formation of TFG-containing cytoplasmic inclusions and concomitant mislocalization of TDP-43 underlie motor neuron degeneration in HMSN-P. Pathological overlap of proteinopathies involving TFG and TDP-43 highlights a new pathway leading to motor neuron degeneration.     

Role of a conserved glycine triplet in the NSS amino acid transporter KAAT1

K(+)-coupled amino acid transporter 1 (KAAT1) belongs to the NSS family of solute transporters and it is expressed in the midgut and in salivary glands of Manduca sexta larvae. As more than 80% of family members, KAAT1 shows a stretch of three glycines (G85-G87) that according to the structure of the prototype transporter LeuT, is located close to the access of the permeation pathway. In this work the role of the triplet has been investigated by alanine and cysteine scanning methods in protein heterologously expressed in Xenopus laevis oocytes. All the mutants were functional but the surface expression level was reduced for G85A and G87A mutants and unaffected for G86A mutant. All presented altered amino acid uptake and transport associated currents in the presence of each of the cations (Na(+), K(+), Li(+)) that can be exploited by the wt. G87A mutant induced increased uncoupled fluxes in the presence of all the cations. Cross-linking studies, performed by the treatment of cysteine mutants with the oxidative complex Cu(II)(1,10-phenanthroline)(3), showed that limiting the flexibility of the region by covalent blockage of position 87, causes a significant reduction of amino acid uptake. Na(+) protected G87C mutant from oxidation, both directly and indirectly. The conserved glycine triplet in KAAT1 plays therefore a complex role that allows initial steps of cation interaction with the transporter.     

A catalytic defect in mitochondrial respiratory chain complex I due to a mutation in NDUFS2 in a patient with Leigh syndrome

In this study, we investigated the pathogenicity of a homozygous Asp446Asn mutation in the NDUFS2 gene of a patient with a mitochondrial respiratory chain complex I deficiency. The clinical, biochemical, and genetic features of the NDUFS2 patient were compared with those of 4 patients with previously identified NDUFS2 mutations. All 5 patients presented with Leigh syndrome. In addition, 3 out of 5 showed hypertrophic cardiomyopathy. Complex I amounts in the patient carrying the Asp446Asn mutation were normal, while the complex I activity was strongly reduced, showing that the NDUFS2 mutation affects complex I enzymatic function. By contrast, the 4 other NDUFS2 patients showed both a reduced amount and activity of complex I. The enzymatic defect in fibroblasts of the patient carrying the Asp446Asn mutation was rescued by transduction of wild type NDUFS2. A 3-D model of the catalytic core of complex I showed that the mutated amino acid residue resides near the coenzyme Q binding pocket. However, the K(M) of complex I for coenzyme Q analogs of the Asp446Asn mutated complex I was similar to the K(M) observed in other complex I defects and in controls. We propose that the mutation interferes with the reduction of coenzyme Q or with the coupling of coenzyme Q reduction with the conformational changes involved in proton pumping of complex I.     

The application of modular protein domains in proteomics

The ability of modular protein domains to independently fold and bind short peptide ligands both in vivo and in vitro has allowed a significant number of protein-protein interaction studies to take advantage of them as affinity and detection reagents. Here, we refer to modular domain based proteomics as "domainomics" to draw attention to the potential of using domains and their motifs as tools in proteomics. In this review we describe core concepts of domainomics, established and emerging technologies, and recent studies by functional category. Accumulation of domain-motif binding data should ultimately provide the foundation for domain-specific interactomes, which will likely reveal the underlying substructure of protein networks as well as the selectivity and plasticity of signal transduction.     

Evaluation of the effectiveness of intrauterine treatment with formosulphathiazole of clinical endometritis in postpartum dairy cows

In cattle, elimination of bacterial contamination from the uterine lumen after parturition is often delayed or compromised, and pathogenic bacteria can persist, causing uterine disease and infertility. The aim of this study was to compare the clinical and bacteriologic recovery following a single intrauterine administration of formosulphatiazole, cephapirin or placebo in cows with clinical endometritis. Cows (n = 80), no less than 28 days postpartum, with clinical endometritis were enrolled in the study. Endometritis was diagnosed by a complete reproductive examination, including rectal palpation, ultrasonography, vaginoscopy and uterine swab. All cows were randomly assigned to receive one of three intrauterine treatments (T0): 2500 mg of formosulphatiazole (Group A); 500 mg of cephapirin (Group B); placebo (4250 mg of propylene glycol; Group C). Cows were examined at the first estrus after treatment or no more than 30 days after (T1). Bacteria isolated were E. coli, A. pyogenes, Pasteurella spp. and Streptococcus spp. After treatment, in Group A and B only 6/30 (20.0%) and 6/24 (25.0%) cows showed a positive bacteriologic culture (P > 0.05), while in Group C the number of positive animals was significantly higher (19/26; 73.1%; P < 0.05). At T0, total clinical scores were similar between the three groups (Group A: 5.84 ± 1.07; Group B: 5.91 ± 1.0; Group C: 5.62 ± 1.17; P > 0.05) and indicative of clinical endometritis. At T1, endometritis scores were significantly lower than those reported before uterine infusion (P < 0.05); however, Group A and B score, 0.4 ± 0.9 and 1.0 ± 2.1, respectively, correspond to no and slight endometritis, while animals in Group C reported a total endometritis score significantly higher (4.6 ± 3.5; P < 0.05) corresponding to endometritis. In the present study, a commercial formosulphatiazole preparation was as effective as cephapirin and more effective than placebo for the treatment of clinical endometritis.     

Characterization of an Endo-Processive-Type Xyloglucanase Having a β-1,4-Glucan-Binding Module and an Endo-Type Xyloglucanase from Streptomyces avermitilis

We cloned two glycoside hydrolase family 74 genes, the sav_1856 gene and the sav_2574 gene, from Streptomyces avermitilis NBRC14893 and characterized the resultant recombinant proteins. The sav_1856 gene product (SaGH74A) consisted of a catalytic domain and a family 2 carbohydrate-binding module at the C terminus, while the sav_2574 gene product (SaGH74B) consisted of only a catalytic domain. SaGH74A and SaGH74B were expressed successfully and had molecular masses of 92 and 78 kDa, respectively. Both recombinant proteins were xyloglucanases. SaGH74A had optimal activity at 60°C and pH 5.5, while SaGH74B had optimal activity at 55°C and pH 6.0. SaGH74A was stable over a broad pH range (pH 4.5 to 9.0), whereas SaGH74B was stable over a relatively narrow pH range (pH 6.0 to 6.5). Analysis of the hydrolysis products of tamarind xyloglucan and xyloglucan-derived oligosaccharides indicated that SaGH74A was endo-processive, while SaGH74B was a typical endo-enzyme. The C terminus of SaGH74A, which was annotated as a carbohydrate-binding module, bound to β-1,4-linked glucan-containing soluble polysaccharides such as hydroxyethyl cellulose, barley glucan, and xyloglucan.     

Relationship between glycated hemoglobin and glucose concentrations in the adult Galician population: selection of optimal glycated hemoglobin cut-off points as a diagnostic tool of diabetes mellitus

Aims/hypothesisTo analyze the relationship between glucose and glycated hemoglobin (HbA_1c) in the adult Galician population, evaluate the use of HbA_1c for the screening and diagnosis of diabetes, and calculate the diagnostic threshold required for this purpose.MethodsWe analyzed data on 2848 subjects (aged 18–85 years) drawn from a study undertaken in 2004 to assess the prevalence of diabetes in Galicia. For study purposes, diabetes was defined using the criteria recommended in 2002. Participants were classified into four glucose-based groups. The relationship between glucose and HbA_1c was described using linear regression models, generalized additive models and Spearman's correlation. Diagnostic capacity was assessed, and optimal HbA_1c cut-off points were calculated as a diabetes marker using the receiver operating characteristic curve.ResultsPrevalence of pre-diabetes, unknown diabetes and known diabetes was 20.86, 3.37 and 4.39%, respectively. The correlations between HbA_1c and fasting glucose were higher than those obtained for HbA_1c and glycemia at 2 h of the oral glucose overload (0.344 and 0.270, respectively). Taking glucose levels as the gold standard, a greater discriminatory capacity was obtained for HbA_1c (area under de cruve: 0.839, 95% confidence intervals: 0.788–0.890). Based on the study criteria, the optimal minimum and maximum HbA_1c values were 5.9% and 6.7%, respectively.Conclusions/interpretationHbA_1c did not prove superior to glycemia for diagnosis of diabetes in the adult Galician population, and cannot therefore be used to replace the oral glucose tolerance test for screening and diagnosis purposes. Indeed, determination of glucose is essential to verify the diagnosis in the majority of cases.