In vivo analysis of Saccharomyces cerevisiae plasma membrane ATPase Pma1p isoforms with increased in vitro H+/ATP stoichiometry

Plasma membrane H⁺-ATPase isoforms with increased H⁺/ATP ratios represent a desirable asset in yeast metabolic engineering. In vivo proton coupling of two previously reported Pma1p isoforms (Ser800Ala, Glu803Gln) with increased in vitro H⁺/ATP stoichiometries was analysed by measuring biomass yields of anaerobic maltose-limited chemostat cultures expressing only the different PMA1 alleles. In vivo H⁺/ATP stoichiometries of wildtype Pma1p and the two isoforms did not differ significantly.     

Implication of C-type natriuretic peptide-3 signaling in glycosaminoglycan synthesis and chondrocyte hypertrophy during TGF-β1 induced chondrogenic differentiation of chicken bone marrow-derived mesenchymal stem cells

This study investigated the involvement of CNP-3, chick homologue for human C-type natriuretic peptide (CNP), in TGF-β1 induced chondrogenic differentiation of chicken bone marrow-derived mesenchymal stem cells (MSCs). Chondrogenic differentiation of MSCs in pellet cultures was induced by TGF-β1. Chondrogenic differentiation and glycosaminoglycan synthesis were analyzed on the basis of basic histology, collagen type II expression, and Alcian blue staining. Antibodies against CNP and NPR-B were used to block their function during these processes. Results revealed that expression of CNP-3 and NPR-B in MSCs were regulated by TGF-β1 in monolayer cultures at mRNA level. In pellet cultures of MSCs, TGF-β1 successfully induced chondrogenic differentiation and glycosaminoglycan synthesis. Addition of CNP into the TGF-β1 supplemented chondrogenic differentiation medium further induced the glycosaminoglycan synthesis and hypertrophy of differentiated chondrocytes in these pellets. Pellets induced with TGF-β1 and treated with antibodies against CNP and NPR-B, did show collagen type II expression, however, Alcian blue staining showing glycosaminoglycan synthesis was significantly suppressed. In conclusion, CNP-3/NPR-B signaling may strongly be involved in synthesis of glycosaminoglycans of the chondrogenic matrix and hypertrophy of differentiated chondrocytes during TGF-β1 induced chondrogenic differentiation of MSCs.     

A publish-subscribe model of genetic networks

We present a simple model of genetic regulatory networks in which regulatory connections among genes are mediated by a limited number of signaling molecules. Each gene in our model produces (publishes) a single gene product, which regulates the expression of other genes by binding to regulatory regions that correspond (subscribe) to that product. We explore the consequences of this publish-subscribe model of regulation for the properties of single networks and for the evolution of populations of networks. Degree distributions of randomly constructed networks, particularly multimodal in-degree distributions, which depend on the length of the regulatory sequences and the number of possible gene products, differed from simpler Boolean NK models. In simulated evolution of populations of networks, single mutations in regulatory or coding regions resulted in multiple changes in regulatory connections among genes, or alternatively in neutral change that had no effect on phenotype. This resulted in remarkable evolvability in both number and length of attractors, leading to evolved networks far beyond the expectation of these measures based on random distributions. Surprisingly, this rapid evolution was not accompanied by changes in degree distribution; degree distribution in the evolved networks was not substantially different from that of randomly generated networks. The publish-subscribe model also allows exogenous gene products to create an environment, which may be noisy or stable, in which dynamic behavior occurs. In simulations, networks were able to evolve moderate levels of both mutational and environmental robustness.     

Rapsyn Mutations in Humans Cause Endplate Acetylcholine-Receptor Deficiency and Myasthenic Syndrome

Congenital myasthenic syndromes (CMSs) stem from genetic defects in endplate (EP)-specific presynaptic, synaptic, and postsynaptic proteins. The postsynaptic CMSs identified to date stem from a deficiency or kinetic abnormality of the acetylcholine receptor (AChR). All CMSs with a kinetic abnormality of AChR, as well as many CMSs with a deficiency of AChR, have been traced to mutations in AChR-subunit genes. However, in a subset of patients with EP AChR deficiency, the genetic defect has remained elusive. Rapsyn, a 43-kDa postsynaptic protein, plays an essential role in the clustering of AChR at the EP. Seven tetratricopeptide repeats (TPRs) of rapsyn subserve self-association, a coiled-coil domain binds to AChR, and a RING-H2 domain associates with beta-dystroglycan and links rapsyn to the subsynaptic cytoskeleton. Rapsyn self-association precedes recruitment of AChR to rapsyn clusters. In four patients with EP AChR deficiency but with no mutations in AChR subunits, we identify three recessive rapsyn mutations: one patient carries L14P in TPR1 and N88K in TPR3; two are homozygous for N88K; and one carries N88K and 553ins5, which frameshifts in TPR5. EP studies in each case show decreased staining for rapsyn and AChR, as well as impaired postsynaptic morphological development. Expression studies in HEK cells indicate that none of the mutations hinders rapsyn self-association but that all three diminish coclustering of AChR with rapsyn.     

Coupling between Catalytic Loop Motions and Enzyme Global Dynamics

Catalytic loop motions facilitate substrate recognition and binding in many enzymes. While these motions appear to be highly flexible, their functional significance suggests that structure-encoded preferences may play a role in selecting particular mechanisms of motions. We performed an extensive study on a set of enzymes to assess whether the collective/global dynamics, as predicted by elastic network models (ENMs), facilitates or even defines the local motions undergone by functional loops. Our dataset includes a total of 117 crystal structures for ten enzymes of different sizes and oligomerization states. Each enzyme contains a specific functional/catalytic loop (10–21 residues long) that closes over the active site during catalysis. Principal component analysis (PCA) of the available crystal structures (including apo and ligand-bound forms) for each enzyme revealed the dominant conformational changes taking place in these loops upon substrate binding. These experimentally observed loop reconfigurations are shown to be predominantly driven by energetically favored modes of motion intrinsically accessible to the enzyme in the absence of its substrate. The analysis suggests that robust global modes cooperatively defined by the overall enzyme architecture also entail local components that assist in suitable opening/closure of the catalytic loop over the active site.     

Effect of statins on coronary blood flow after percutaneous coronary intervention in patients with stable coronary artery disease

Aims

Statins have favourable effects on the vascular system. However, few data are available regarding the effect of these drugs on patients undergoing percutaneous coronary intervention (PCI). We sought to determine the impact of prior statin use on coronary blood flow after PCI in patients with stable coronary artery disease (CAD) by using the corrected thrombolysis in myocardial infarction (TIMI) frame count (CTFC).

Methods

A total of 80 consecutive eligible patients (mean age: 60 ± 7 years, 65?% male) with the diagnosis of stable CAD who were hospitalised for elective PCI were retrospectively enrolled in our study. The study population was divided into two groups according to statin use at least 6 months before PCI. Group 1 comprised of 51 patients (67?% male; mean age: 58 ± 4 years) taking statins and group 2 comprised of 29 patients (62?% male; mean age: 60 ± 3 years) not taking statins. PCI was applied to de novo type A lesions. CTFC was calculated for the treated vessels at baseline and after PCI.

Results

The two groups had similar characteristics in terms of age, sex, concomitant medications, lesion characteristics, pre-procedural CTFC, lipid parameters, and risk factors for CAD. Post-PCI CTFC (16 ± 3 vs. 22 ± 5, p = 0.01) and hs-CRP (2.1 ± 0.7 mg/l vs. 6.1 ± 2 mg/l, p = 0.01) in patients receiving statins before PCI were significantly lower than in patients without statin therapy. Multiple logistic regression analysis showed that statin pre-treatment (OR 2.5, 95?% CI 1.2 to 3.8, p < 0.001) and hs-CRP level (OR 1.8, 95?% CI 1.2 to 2.4, p = 0.001) were independent predictors of post-PCI CTFC.

Conclusions

In patients with stable CAD undergoing PCI, receipt of long-term statin therapy was associated with improvement in epicardial perfusion after PCI.