Comparison of the reaction progress of calcineurin with Mn2+ and Mg2+

Activation of calcineurin by Mn2+ and Mg2+ was compared using a heavy atom isotope analogue of the substrate p-nitrophenyl phosphate (pNPP). Heavy atom isotope effects were measured for Mg2+ activation and compared to published results of the isotope effects with Mn2+ as the activating metal. Isotope effects were measured for the kinetic parameter Vmax/Km at the nonbridging oxygen atoms [18(V/K)nonbridge]; at the position of bond cleavage in the bridging oxygen atom [18(V/K)bridge]; and at the nitrogen atom in the nitrophenol leaving group [15(V/K)]. The isotope effects increased in magnitude upon changing from an optimal pH to a nonoptimal pH; the 18(V/K)bridge effect increased from 1.0154 (+/-0.0007) to 1.0198 (+/-0.0002), and the 15(V/K) effect increased from 1.0018 (+/-0. 0002) to 1.0021 (+/-0.0003). The value for 18(V/K)nonbridge is 0. 9910 (+/-0.0003) at pH 7.0. As with Mn2+, the 18(V/K)nonbridge isotope effect indicated that the dianion was the substrate for catalysis, and that a dissociative transition state was operative for the phosphoryl transfer. Comparison to results for Mn2+ activation suggested that chemistry was more rate-limiting with Mg2+ than with Mn2+. Changing the activating metal concentration showed opposite trends with increasing Mg2+ increasing the commitment factor and seemingly making the chemistry less rate-limiting. The influence of viscosity was evaluated as well to gauge the role of chemistry. The activation of calcineurin-catalyzed hydrolysis of pNPP1 by Mg2+ or Mn2+ at pH 7.0 was compared in the presence of viscogens, glycerol and poly(ethylene glycol). Increasing glycerol caused different effects with the two activators. With Mn2+ as the activator, calcineurin activity showed a normal response with kcat and kcat/Km decreasing with viscosity. There was an inverse response with Mg2+ as the activator as values of kcat/Km increased with viscosity. From values of the normalized kcat/Km with Mn2+, the chemistry was found to be partially rate-limiting, consistent with previous heavy atom isotope studies (22). The effect observed for Mg2+ seems consistent with a change in the rate-limiting step for the two different metals at pH 7.0.     

Small marker chromosomes in two patients with segmental aneusomy for proximal 17p

We report a nine-year-old girl (patient 1934) and a five-year-old boy (patient 2170) with small, de novo supernumerary marker chromosomes (SMCs) derived from proximal 17p. The clinical features of patient 1934 include developmental delay, triangular face, prominent forehead, low set ears, dental abnormalities, a high arched palate, long, flexible fingers, and joint laxity. Patient 2170 is affected with developmental delay, oral-motor dyspraxia/verbal apraxia, thick upper and lower lips, bilateral fifth finger clinodactyly, joint laxity and mild hypotonia. G-banded chromosome analysis of patient 1934 revealed mosaicism for a SMC in 72% of peripheral lymphocytes analyzed, whereas analysis of patient 2170 identified a smaller SMC present in 100% of cells analyzed. Fluorescence in situ hybridization (FISH) studies demonstrated that both of the SMCs derived from 17p10-p11.2. Using FISH and array-CGH analysis, the proximal breakpoints mapped within the centromere and the distal breakpoints were both located within the Smith-Magenis syndrome (SMS) common deletion region. We compare the clinical characteristics of our patients with those previously reported to have either SMC including 17p or duplications of proximal 17p in an effort to further delineate the phenotype of trisomy 17p10-p11.2 and to elucidate genotype-phenotype correlations.     

Analysis of superfamily specific profile-profile recognition accuracy

Background  

Annotation of sequences that share little similarity to sequences of known function remains a major obstacle in genome annotation. Some of the best methods of detecting remote relationships between protein sequences are based on matching sequence profiles. We analyse the superfamily specific performance of sequence profile-profile matching. Our benchmark consists of a set of 16 protein superfamilies that are highly diverse at the sequence level. We relate the performance to the number of sequences in the profiles, the profile diversity and the extent of structural conservation in the superfamily.     

Production of functional single-chain Fv antibodies in the cytoplasm of Escherichia coli

Production of intracellular antibodies in Escherichia coli has been thought unlikely owing to an inability to form stable disulfide bonds in the cytoplasm, a necessary step in the folding of most immunoglobulin (Ig) domains. This work investigates whether E. coli strains carrying mutations in the major intracellular disulfide bond-reduction systems (i.e. the thioredoxin and the glutathione/glutaredoxin pathways) allow the oxidation and folding of single chain variable fragment (scFv) antibodies in the cytoplasm. The effect of the co-expression of disulfide bond chaperones in these cells was also examined. An scFv that recognizes the alternative sigma factor sigma(54) was used as a model to investigate disulfide bond formation and the folding of Ig domains in E. coli. The results demonstrate that functional intrabodies, with oxidized disulfide bonds in their Ig domains, are produced efficiently in E. coli cells carrying mutations in the glutathione oxidoreductase (gor) and the thioredoxin reductase (trxB) genes and co-expressing a signal-sequence-less derivative of the disulfide-bond isomerase DsbC ((Delta)ssDsbC). We obtained evidence indicating that (Delta)ssDsbC acts as a chaperone promoting the correct folding and oxidation of scFvs.     

Diagnostic criteria for diabetes revisited: making use of combined criteria

Background  

Existing cut-offs for fasting plasma glucose (FPG) and post-load glucose (2hPG) criteria are not equivalent in the diagnosis of diabetes and glucose intolerance. Adjusting cut-offs of single measurements have not helped so we undertook this project to see if they could be complementary.     

Inflammatory cells induce neointimal growth in a rat arterial autograft model

Subendothelial invasion by leukocytes is a sign of intimal thickening in arteriosclerosis and in the response of a vessel to mechanical damage. Our study was designed to establish whether these cells are implicated in the formation of a neointima in an autologous arterial graft model in the rat and to evaluate the effects of cyclosporin A (CsA). Three study groups were established according to whether the animals were treated with CsA-Cp (Sandimmun), CsA-Et (ethanol vehicle) or received no treatment (control group). Both drug forms were administered (5 mg/kg/day, s.c.) from 4 days prior to surgery until the time of sacrifice. Antibodies specific for lymphocytes (CD4, CD8), monocytes/macrophages-ED1, smooth muscle alpha-actin and the von Willebrand factor (vWF) were used to identify the cells in the grafted arterial wall. In control grafts, the neointima had formed by 2 weeks post-implant. However, the cells comprising this layer generally presented no positivity whatsoever towards the antibodies employed. At 50 days, the new layer was observed to be formed by a vWF-positive endothelium and alpha-actin-positive cells. In all three groups, several polymorphonuclear (PMN) cells adhered to the denuded luminal surface from 7 days onwards. In the treated animals, neutrophils and monocytes were seen to infiltrate intimal and medial layers during the later post-implant stages. Around the third week post-implant, the neointima had reached the grafted segment from the distal portion of the recipient artery, and by 50 days it was similar to that seen in control specimens. Our findings suggest that: a) neutrophils play a role in neointimal thickening in this arterial autograft model; and b) CsA promotes the adhesion and infiltration of neutrophils in the injured arterial wall.     

Evaluation of the smooth muscle cell component and apoptosis in the varicose vein wall

This study was designed to evaluate the role of the smooth muscle cell and the apoptosis in the pathogenesis of the varicose vein. Segments of saphenous vein were obtained from healthy subjects and from those with varicose veins. The vein specimens were subdivided according to subject age (younger or older than 50 years) and according to the varicose vein source (distal or proximal). Morphological, ultrastructural, cell proliferation (anti-PCNA method) and cell death (TUNEL method) analysis were performed. The walls of healthy, control vein specimens acquired a more collagenous and papillomatous appearance with age. A slight increase in the number of TUNEL-positive cells was also observed in specimens from older subjects. The proportion of apoptotic cells was much greater in the varicose veins than in control specimens. Most cellular alterations were seen in proximal varicose segments obtained from young subjects. These specimens showed hypertrophic areas with a high degree of cellularity (both in the media and in the thickened intima). The highest proportion of apoptotic cells and collagenisation were also observed in these areas. The enhanced number of apoptotic cells in varicose veins observed mainly in proximal/young vein specimens could be responsible, at least in part, for the acceleration of the final fibrosclerotic process characteristic of the varicose vein wall.