Free and polymerized tubulin in cultured bone cells and Chinese hamster ovary cells: the influence of cold and hormones

A low pH method of liposome-membrane fusion (Schneider et al., 1980, Proc. Natl. Acad. Sci. U. S. A. 77:442) was used to enrich the mitochondrial inner membrane lipid bilayer 30-700% with exogenous phospholipid and cholesterol. By varying the phospholipid-to- cholesterol ratio of the liposomes it was possible to incorporate specific amounts of cholesterol (up to 44 mol %) into the inner membrane bilayer in a controlled fashion. The membrane surface area increased proportionally to the increase in total membrane bilayer lipid. Inner membrane enriched with phospholipid only, or with phospholipid plus cholesterol up to 20 mol %, showed randomly distributed intramembrane particles (integral proteins) in the membrane plane, and the average distance between intramembrane particles increased proportionally to the amount of newly incorporated lipid. Membranes containing between 20 and 27 mol % cholesterol exhibited small clusters of intramembrane particles while cholesterol contents above 27 mol % resulted in larger aggregations of intramembrane particles. In phospholipid-enriched membranes with randomly dispersed intramembrane particles, electron transfer activities from NADH- and succinate-dehydrogenase to cytochrome c decreased proportionally to the increase in distance between the particles. In contrast, these electron- transfer activities increased with decreasing distances between intramembrane particles brought about by cholesterol incorporation. These results indicate that (a) catalytically interacting redox components in the mitochondrial inner membrane such as the dehydrogenase complexes, ubiquinone, and heme proteins are independent, laterally diffusible components; (b) the average distance between these redox components is effected by the available surface area of the membrane lipid bilayer; and (c) the distance over which redox components diffuse before collision and electron transfer mediates the rate of such transfer.     

Tomato mutants altered in bacterial disease resistance provide evidence for a new locus controlling pathogen recognition.

We have employed a genetic approach to study the resistance of tomato to the phytopathogenic bacterium Pseudomonas syringae pv tomato. Resistance to P. s. tomato depends upon expression of the Pto locus in tomato, which encodes a protein with similarity to serine/threonine protein kinases and recognizes pathogen strains expressing the avirulence gene avrPto. Eleven tomato mutants were isolated with altered resistance to P. s. tomato strains expressing avrPto. We identified mutations both in the Pto resistance locus and in a new locus designated Prf (for Pseudomonas resistance and fenthion sensitivity). The genetic approach allowed us to dissect the roles of these loci in signal transduction in response to pathogen attack. Lines carrying mutations in the Pto locus vary 200-fold in the degree to which they are susceptible to P. s. tomato strains expressing avrPto. The pto mutants retain sensitivity to the organophosphate insecticide fenthion; this trait segregates with Pto in genetic crosses. This result suggested that contrary to previous hypotheses, the Pto locus controls pathogen recognition but not fenthion sensitivity. Interestingly, mutations in the prf locus result in both complete susceptibility to P. s. tomato and insensitivity to fenthion, suggesting that Prf plays a role in tomato signaling in response to both pathogen elicitors and fenthion. Because pto and prf mutations do not alter recognition of Xanthomonas campestris strains expressing avrBsP, an avirulence gene recognized by all tested tomato cultivars, Prf does not play a general role in disease resistance but possibly functions specifically in resistance against P. s. tomato. Genetic analysis of F2 populations from crosses of pto and prf homozygotes indicated that the Pto and Prf loci are tightly linked.     

Evolutionary dynamics of the kinetochore network in eukaryotes as revealed by comparative genomics

During eukaryotic cell division, the sister chromatids of duplicated chromosomes are pulled apart by microtubules, which connect via kinetochores. The kinetochore is a multiprotein structure that links centromeres to microtubules, and that emits molecular signals in order to safeguard the equal distribution of duplicated chromosomes over daughter cells. Although microtubule‐mediated chromosome segregation is evolutionary conserved, kinetochore compositions seem to have diverged. To systematically inventory kinetochore diversity and to reconstruct its evolution, we determined orthologs of 70 kinetochore proteins in 90 phylogenetically diverse eukaryotes. The resulting ortholog sets imply that the last eukaryotic common ancestor (LECA) possessed a complex kinetochore and highlight that current‐day kinetochores differ substantially. These kinetochores diverged through gene loss, duplication, and, less frequently, invention and displacement. Various kinetochore components co‐evolved with one another, albeit in different manners. These co‐evolutionary patterns improve our understanding of kinetochore function and evolution, which we illustrated with the RZZ complex, TRIP13, the MCC, and some nuclear pore proteins. The extensive diversity of kinetochore compositions in eukaryotes poses numerous questions regarding evolutionary flexibility of essential cellular functions.     

Dynamic modification of a mutant cytoplasmic cysteine residue modulates the conductance of the human 5-HT3A receptor

Structural models suggest that Arg(436) lies within five cytoplasmic portals of the 5-HT(3A) receptor. We tested both the accessibility of residue 436 and the influence of its charge on single channel conductance (gamma) by substituting Arg(436) with Cys and examining the effect of methanethiosulfonate (MTS) reagents on gamma. Inclusion of positively charged 2-aminoethyl-MTS (MTSEA) within the electrode solution reduced gamma of 5-HT(3A)(R436C) receptors in outside-out patches from 7.8 +/- 0.5 to 5.0 +/- 0.5 picosiemens (pS). To increase gamma, we substituted Arg(436) by Cys in the 5-HT(3A)(R432Q,R440A) mutant, yielding the 5-HT(3A)(QCA) construct with a gamma of 17.7 +/- 0.4 pS. Modification of 5-HT(3A)(QCA) receptors by MTSEA or 2-(trimethylammonium) ethyl-MTS reduced gamma to 8.7 +/- 0.5 and 6.7 +/- 0.4 pS, respectively, both significantly below that of channels exposed to nonpolar propyl-MTS. Extracellular MTSEA, but not 2-(trimethylammonium) ethyl-MTS, crossed the membrane and in so doing slowly (tau = 94 s) reduced gamma. MTSEA more rapidly (tau = 15 s) reduced the gamma of 5-HT(3A)(QCA) receptors in inside-out patches, an effect reversed by the reducing agent dithiothreitol. Cys(436) modification by negatively charged 2-carboxyethyl-MTS and 2-sulfonatoethyl-MTS increasedgamma to 23 +/- 1.0 and 26 +/- 0.7 pS, respectively. MTS reagents did not affect gamma values for 5-HT(3A)(QDA) constructs with Cys substituted for Lys(431) predicted to be outside the entrance to the portals. Collectively, the data demonstrate that the dynamic modification of the charge of a cytoplasmic residue regulates gamma, consistent with the existence of cytoplasmic portals that impose a rate-limiting barrier to ion conduction in Cys loop receptors.     

Substituted 9-aminoacridine-4-carboxamides tethered to platinum(II)diamine complexes: Chemistry, cytotoxicity and DNA sequence selectivity

Three platinum complexes in which substituted (7-OMe, 9-NH₂; 7-F, 9-NH₂; and 7-H, 9-NH(CH₂)₂OH) 9-aminoacridine-4-carboxamides were tethered to a platinum(II)diamine moiety were synthesised and characterised at the chemical and biological level. These variants showed a decrease in cytotoxicity, as measured by IC₅₀ values in HeLa cells, when compared with the parent 7-H, 9-NH₂ compound. The 7-F and 9-NH(CH₂)₂OH substituents gave rise to a small decrease in cytotoxicity, and the 7-OMe substituent resulted in a larger decrease in cytotoxicity. Their binding to purified pUC19 plasmid DNA was investigated and it was found that the addition of 7-F, 9-NH(CH₂)₂OH and especially the 7-OMe substituents, resulted in reduced DNA binding. This correlated well with the IC₅₀ cytotoxicity values. However, the DNA sequence selectivity was unaffected by the addition of these moieties.     

Modeling <Emphasis Type="Italic">Neisseria meningitidis</Emphasis> metabolism: from genome to metabolic fluxes

Background  

Neisseria meningitidis is a human pathogen that can infect diverse sites within the human host. The major diseases caused by N. meningitidis are responsible for death and disability, especially in young infants. In general, most of the recent work on N. meningitidis focuses on potential antigens and their functions, immunogenicity, and pathogenicity mechanisms. Very little work has been carried out on Neisseria primary metabolism over the past 25 years.     

Simulation of human atherosclerotic femoral plaque tissue: the influence of plaque material model on numerical results

Background

Due to the limited number of experimental studies that mechanically characterise human atherosclerotic plaque tissue from the femoral arteries, a recent trend has emerged in current literature whereby one set of material data based on aortic plaque tissue is employed to numerically represent diseased femoral artery tissue. This study aims to generate novel vessel-appropriate material models for femoral plaque tissue and assess the influence of using material models based on experimental data generated from aortic plaque testing to represent diseased femoral arterial tissue.

Methods

Novel material models based on experimental data generated from testing of atherosclerotic femoral artery tissue are developed and a computational analysis of the revascularisation of a quarter model idealised diseased femoral artery from a 90% diameter stenosis to a 10% diameter stenosis is performed using these novel material models. The simulation is also performed using material models based on experimental data obtained from aortic plaque testing in order to examine the effect of employing vessel appropriate material models versus those currently employed in literature to represent femoral plaque tissue.

Results

Simulations that employ material models based on atherosclerotic aortic tissue exhibit much higher maximum principal stresses within the plaque than simulations that employ material models based on atherosclerotic femoral tissue. Specifically, employing a material model based on calcified aortic tissue, instead of one based on heavily calcified femoral tissue, to represent diseased femoral arterial vessels results in a 487 fold increase in maximum principal stress within the plaque at a depth of 0.8 mm from the lumen.

Conclusions

Large differences are induced on numerical results as a consequence of employing material models based on aortic plaque, in place of material models based on femoral plaque, to represent a diseased femoral vessel. Due to these large discrepancies, future studies should seek to employ vessel-appropriate material models to simulate the response of diseased femoral tissue in order to obtain the most accurate numerical results.

     

Review and classification of variability analysis techniques with clinical applications

Background

Representation of independent biophysical sources using Fourier analysis can be inefficient because the basis is sinusoidal and general. When complex fractionated atrial electrograms (CFAE) are acquired during atrial fibrillation (AF), the electrogram morphology depends on the mix of distinct nonsinusoidal generators. Identification of these generators using efficient methods of representation and comparison would be useful for targeting catheter ablation sites to prevent arrhythmia reinduction.

Method

A data-driven basis and transform is described which utilizes the ensemble average of signal segments to identify and distinguish CFAE morphologic components and frequencies. Calculation of the dominant frequency (DF) of actual CFAE, and identification of simulated independent generator frequencies and morphologies embedded in CFAE, is done using a total of 216 recordings from 10 paroxysmal and 10 persistent AF patients. The transform is tested versus Fourier analysis to detect spectral components in the presence of phase noise and interference. Correspondence is shown between ensemble basis vectors of highest power and corresponding synthetic drivers embedded in CFAE.

Results

The ensemble basis is orthogonal, and efficient for representation of CFAE components as compared with Fourier analysis (p ≤ 0.002). When three synthetic drivers with additive phase noise and interference were decomposed, the top three peaks in the ensemble power spectrum corresponded to the driver frequencies more closely as compared with top Fourier power spectrum peaks (p ≤ 0.005). The synthesized drivers with phase noise and interference were extractable from their corresponding ensemble basis with a mean error of less than 10%.

Conclusions

The new transform is able to efficiently identify CFAE features using DF calculation and by discerning morphologic differences. Unlike the Fourier transform method, it does not distort CFAE signals prior to analysis, and is relatively robust to jitter in periodic events. Thus the ensemble method can provide a useful alternative for quantitative characterization of CFAE during clinical study.