A genomewide exploration suggests a new candidate gene at chromosome 11q23 as the major determinant of plasma homocysteine levels: results from the GAIT project

Homocysteine (Hcy) plasma level is an independent risk marker for venous thrombosis, myocardial infarction, stroke, congestive heart failure, osteoporotic fractures, and Alzheimer disease. Hcy levels are determined by the interaction of genetic and environmental factors. The genetic basis is still poorly understood, since only the MTHFR 677 C-->T polymorphism has been consistently associated with plasma Hcy levels. We conducted a genomewide linkage scan for genes affecting variation in plasma Hcy levels in 398 subjects from 21 extended Spanish families. A variance-components linkage method was used to analyze the data. The strongest linkage signal (LOD score of 3.01; genomewide P = .035) was found on chromosome 11q23, near marker D11S908, where a candidate gene involved in the metabolism of Hcy (the nicotinamide N-methyltransferase gene [NNMT]) is mapped. Haplotype analyses of 10 single-nucleotide polymorphisms within this gene found one haplotype associated with plasma Hcy levels (P = .0003). Our results, to our knowledge, represent the first genomic scan for quantitative variation in Hcy plasma levels. They strongly suggest that the NNMT gene could be a major genetic determinant of plasma Hcy levels in Spanish families. Since this gene encodes an enzyme involved in Hcy synthesis, this finding would be consistent with known biochemical pathways. These data could be relevant in determining the relationships between Hcy level, cardiovascular disease, osteoporosis, and Alzheimer disease.     

Mechanism of anionic conduction across ClC

ClC chloride channels are voltage-gated transmembrane proteins that have been associated with a wide range of regulatory roles in vertebrates. To accomplish their function, they allow small inorganic anions to efficiently pass through, while blocking the passage of all other particles. Understanding the conduction mechanism of ClC has been the subject of many experimental investigations, but until now, the detailed dynamic mechanism was not known despite the availability of crystallographic structures. We investigate Cl(-) conduction by means of an all-atom molecular dynamics simulation of the ClC channel in a membrane environment. Based on our simulation results, we propose a king-of-the-hill mechanism for permeation, in which a lone ion bound to the center of the ClC pore is pushed out by a second ion that enters the pore and takes its place. Although the energy required to extract the single central ion from the pore is enormous, by resorting to this two-ion process, the largest free energy barrier for conduction is reduced to 4 kcal/mol. At the narrowest part of the pore, residues Tyr-445 and Ser-107 stabilize the central ion. There, the bound ion blocks the pore, disrupting the formation of a continuous water file that could leak protons, possibly preventing the passage of uncharged solutes.     

Impaired binding affinity of electronegative low-density lipoprotein (LDL) to the LDL receptor is related to nonesterified fatty acids and lysophosphatidylcholine content

The binding characteristics of electropositive [LDL(+)] and electronegative LDL [LDL(-)] subfractions to the LDL receptor (LDLr) were studied. Saturation kinetic studies in cultured human fibroblasts demonstrated that LDL(-) from normolipemic (NL) and familial hypercholesterolemic (FH) subjects had lower binding affinity than their respective LDL(+) fractions (P < 0.05), as indicated by higher dissociation constant (K(D)) values. FH-LDL(+) also showed lower binding affinity (P < 0.05) than NL-LDL(+) (K(D), sorted from lower to higher affinity: NL-LDL(-), 33.0 +/- 24.4 nM; FH-LDL(-), 24.4 +/- 7.1 nM; FH-LDL(+), 16.6 +/- 7.0 nM; NL-LDL(+), 10.9 +/- 5.7 nM). These results were confirmed by binding displacement studies. The impaired affinity binding of LDL(-) could be attributed to altered secondary and tertiary structure of apolipoprotein B, but circular dichroism (CD) and tryptophan fluorescence (TrpF) studies revealed no structural differences between LDL(+) and LDL(-). To ascertain the role of increased nonesterified fatty acids (NEFA) and lysophosphatidylcholine (LPC) content in LDL(-), LDL(+) was enriched in NEFA or hydrolyzed with secretory phospholipase A(2). Modification of LDL gradually decreased the affinity to LDLr in parallel to the increasing content of NEFA and/or LPC. Modified LDLs with a NEFA content similar to that of LDL(-) displayed similar affinity. ApoB structure studies of modified LDLs by CD and TrpF showed no difference compared to LDL(+) or LDL(-). Our results indicate that NEFA loading or phospholipase A(2) lipolysis of LDL leads to changes that affect the affinity of LDL to LDLr with no major effect on apoB structure. Impaired affinity to the LDLr shown by LDL(-) is related to NEFA and/or LPC content rather than to structural differences in apolipoprotein B.     

Oscillometric assessment of airway obstruction in a mechanical model of vocal cord dysfunction

Vocal cord dysfunction (VCD) is characterized by inappropriate adduction of the vocal cords, particularly during inspiration, resulting in obstruction and airflow limitation. Direct visualization of the vocal cords with laryngoscopy is the 'gold standard' for diagnosing VCD. However, it is an invasive technique that may induce airway irritation. The aim of this study was to determine whether the forced oscillation technique (FOT) is useful to estimate the degree of closure of a non-linear orifice under conditions mimicking those found in VCD. The FOT (5 Hz, +/-1 cm H(2)O) was applied to an airway model simultaneously with constant levels of flow in the normal breathing range (0-0.8l/s). Pressure-flow (P(0)-V'(0)) curves, quasi-static resistance (R(eff)) and oscillatory resistance (R(FOT)) were measured in orifices with different areas (0.15-1.12 cm2) and shapes and in an orifice with variable area. Their pressure-flow relationship followed a quadratic model. Changes in R(FOT) normalized by flow (DeltaR(FOT)/V'(0)) were related to changes in the area of the vocal cord model (1/A(VC2)(2)-1/A(VC1)(2)) from maximum aperture (A(VC1)) to different degrees of closure (A(VC2)): DeltaR(FOT)/V'(0)=1.93(1/A(VC2)(2)-1/A(VC1)(2))+2.08 cm H(2)Os(2)/l(2); r(2)=0.99. We conclude that FOT could be a useful tool for non-invasively assessing glottic closure in VCD diagnosis, obviating the need for other invasive techniques.     

A plant type 2 metallothionein (MT) from cork tissue responds to oxidative stress

Expression of plant metallothionein genes has been reported in a variety of senescing tissues, such as leaves and stems, ripening fruits, and wounded tissues, and has been proposed to function in both metal chaperoning and scavenging of reactive oxygen species. In this work, it is shown that MT is also associated with suberization, after identifying a gene actively transcribed in Quercus suber cork cells as a novel MT. This cDNA, isolated from a phellem cDNA library, encodes a MT that belongs to type 2 plant MTs (QsMT). Expression of the QsMT cDNA in E. coli grown in media supplemented with Zn, Cd, or Cu has yielded recombinant QsMT. Characterization of the respective metal aggregates agrees well with a copper-related biological role, consistent with the capacity of QsMT to restore copper tolerance to a MT-deficient, copper-sensitive yeast mutant. Furthermore, in situ hybridization results demonstrate that RNA expression of QsMT is mainly observed under conditions related to oxidative stress, either endogenous, as found in cork or in actively proliferating tissues, or exogenous, for example, in response to H(2)O(2) or paraquat treatments. The putative role of QsMT in oxidative stress, both as a free radical scavenger via its sulphydryl groups or as a copper chelator is discussed.     

Dedifferentiated adult articular chondrocytes: a population of human multipotent primitive cells

OBJECTIVE: To test the hypothesis that dedifferentiated adult human cartilage chondrocytes (HAC) are a true multipotent primitive population. METHODS: Studies to characterize dedifferentiated HAC included cell cycle and quiescence analysis, cell fusion, flow-FISH telomere length assays, and ABC transporter analysis. Dedifferentiated HAC were characterized by flow cytometry, in parallel with bone marrow mesenchymal stem cells (MSC) and processed lipoaspirate (PLA) cells. The in vitro differentiation potential of dedifferentiated HAC was studied by cell culture under several inducing conditions, in multiclonal and clonal cell populations. RESULTS: Long-term HAC cultures were chromosomically stable and maintained cell cycle dynamics while showing telomere shortening. The phenotype of dedifferentiated HAC was quite similar to that of human bone marrow MSC. In addition, this population expressed human embryonic stem cell markers. Multiclonal populations of dedifferentiated HAC differentiated to chondrogenic, osteogenic, adipogenic, myogenic, and neurogenic lineages. Following VEGF induction, dedifferentiated HAC expressed characteristics of endothelial cells, including AcLDL uptake. A total of 53 clonal populations of dedifferentiated HAC were efficiently expanded; 17 were able to differentiate to chondrogenic, osteogenic, and adipogenic lineages. No correlation was observed between telomere length or quiescent population and differentiation potential in the clones assayed. CONCLUSION: Dedifferentiated HAC should be considered a human multipotent primitive population.     

Fine-tuning the hydrophobicity of a mitochondria-targeted antioxidant

The mitochondria-targeted antioxidant MitoQ comprises a ubiquinol moiety covalently attached through an aliphatic carbon chain to the lipophilic triphenylphosphonium cation. This cation drives the membrane potential-dependent accumulation of MitoQ into mitochondria, enabling the ubiquinol antioxidant to prevent mitochondrial oxidative damage far more effectively than untargeted antioxidants. We sought to fine-tune the hydrophobicity of MitoQ so as to control the extent of its membrane binding and penetration into the phospholipid bilayer, and thereby regulate its partitioning between the membrane and aqueous phases within mitochondria and cells. To do this, MitoQ variants with 3, 5, 10 and 15 carbon aliphatic chains were synthesised. These molecules had a wide range of hydrophobicities with octan-1-ol/phosphate buffered saline partition coefficients from 2.8 to 20000. All MitoQ variants were accumulated into mitochondria driven by the membrane potential, but their binding to phospholipid bilayers varied from negligible for MitoQ3 to essentially total for MitoQ15. Despite the span of hydrophobicites, all MitoQ variants were effective antioxidants. Therefore, it is possible to fine-tune the degree of membrane association of MitoQ and other mitochondria targeted compounds, without losing antioxidant efficacy. This indicates how the uptake and distribution of mitochondria-targeted compounds within mitochondria and cells can be controlled, thereby facilitating investigations of mitochondrial oxidative damage.     

Foot infections associated with Arcanobacterium pyogenes in free-living fallow deer (Dama dama)

We describe foot infection associated with Arcanobacterium pyogenes in three adult male free-living fallow deer (Dama dama) from Sueve Regional Hunting Reserve (Principality of Asturias, Spain). Affected fallow deer were culled in November 1997 and 1998 during the hunting season. Necropsy, radiography, and microbiologic analysis were carried out for each animal. Unilateral swelling of one extremity at the coronary band was observed in all three cases. Areas of bone loss, severe periosteal reaction, and soft tissue swelling were seen on radiography. Lead fragments were observed in one fallow deer. Seven bacterial species were isolated, but only Arcanobacterium pyogenes was routinely found. Weather conditions in the area (mild temperatures and high humidity), the land (alternating pasture land and rock), the animal population density (both fallow deer and domestic herds of cows, horses, sheep, and goats, live side by side in the same areas), and hunting activities could be related to the frequency of these infections.