Organization of the mouse microfibril-associated glycoprotein-2 (MAGP-2) gene

A 1.4-kb EST clone encoding mouse microfibril-associated glycoprotein-2 (MAGP-2), identified by its similarity with the reported human cDNA, was used to screen a mouse 129 genomic bacterial artificial chromosome (BAC) library. The mouse gene contains 10 exons spanning 16 kb, located on the distal region of Chromosome (Chr) 6. The exons range in size from 24 to 963 bp, with the ATG located in exon 2. The tenth and largest exon contains 817 bp of 3′ untranslated sequence, including a B2 repetitive element. Northern analysis demonstrates abundant expression of MAGP-2 mRNA in skeletal muscle, lung, and heart. Sequence analysis of additional cDNA clones suggests that the two mRNA forms of MAGP-2 in the mouse arise from alternative polyadenylation site usage. The promoter does not contain an obvious TATA box, and the sequence surrounding the start site does not conform to the consensus for an initiator promoter element. Additionally, the mouse promoter contains 22 copies of a CT dinucleotide repeat sequence located ∼155 bp 5′ to exon 1. Received: 27 August 1999 / Accepted: 2 November 1999     

Relationship between ion distribution and membrane potential during a steady state

Equations were derived showing the relationship between the membrane potential and the quantities which influence it under steady state conditions. Essentially, the membrane potential is caused by the valence and concentration of the non-permeating ions. The permeating ions can modify the membrane potential by altering the relative concentration of the non-permeating ions with respect to the concentration of the permeating ions. For muscle, the sodium cations act as the non-permeating ions in the extracellular environment by the maintenance of some type of active metabolic process and large anions act as the non-permeating ions in the intracellular environment. Both of these non-permeating ions contribute about equally to the maintenance of the resting membrane potential. When the active metabolic process for sodium extrusion breaks down or when acids are added, the membrane potential should decrease. Water should enter the cell when the sodium metabolic process is diminished; water should leave the cell when acids are added. When acid is added, it is expected that the cations potassium and sodium will leave the cell with little or no shift of the chloride ions.     

Biophysical Analysis of Kindlin-3 Reveals an Elongated Conformation and Maps Integrin Binding to the Membrane-distal ��-Subunit NPXY Motif

Kindlin-3, a 75-kDa protein, has been shown to be critical for hemostasis, immunity, and bone metabolism via its role in integrin activation. The Kindlin family is hallmarked by a FERM domain comprised of F1, F2, and F3 subdomains together with an N-terminal F0 domain and a pleckstrin homology domain inserted in the F2 domain. Recombinant Kindlin-3 was cloned, expressed, and purified, and its domain organization was studied by x-ray scattering and other techniques to reveal an extended conformation. This unusual elongated structure is similar to that found in the paralogue Talin head domain. Analytical ultracentrifugation experiments indicated that Kindlin-3 forms a ternary complex with the Talin and β-integrin cytoplasmic tails. NMR showed that Kindlin-3 specifically recognizes the membrane-distal tail NPXY motif in both the β_1A and β_1D isoforms, although the interaction is stronger with β_1A. An upstream Ser/Thr cluster in the tails also plays a critical role. Overall these data support current biological, clinical, and mutational data on Kindlin-3/β-tail binding and provide novel insights into the overall conformation and interactions of Kindlin-3.     

Hypothesis tests for stratified mark‐specific proportional hazards models with missing covariates,with application to HIV vaccine efficacy trials

This article develops hypothesis testing procedures for the stratified mark‐specific proportional hazards model with missing covariates where the baseline functions may vary with strata. The mark‐specific proportional hazards model has been studied to evaluate mark‐specific relative risks where the mark is the genetic distance of an infecting HIV sequence to an HIV sequence represented inside the vaccine. This research is motivated by analyzing the RV144 phase 3 HIV vaccine efficacy trial, to understand associations of immune response biomarkers on the mark‐specific hazard of HIV infection, where the biomarkers are sampled via a two‐phase sampling nested case‐control design. We test whether the mark‐specific relative risks are unity and how they change with the mark. The developed procedures enable assessment of whether risk of HIV infection with HIV variants close or far from the vaccine sequence are modified by immune responses induced by the HIV vaccine; this question is interesting because vaccine protection occurs through immune responses directed at specific HIV sequences. The test statistics are constructed based on augmented inverse probability weighted complete‐case estimators. The asymptotic properties and finite‐sample performances of the testing procedures are investigated, demonstrating double‐robustness and effectiveness of the predictive auxiliaries to recover efficiency. The finite‐sample performance of the proposed tests are examined through a comprehensive simulation study. The methods are applied to the RV144 trial.     

Charge immobilization linked to inactivation in the aggregation model of channel gating

Expected gating currents are derived analytically from a continuous, time-homogenous Markov process formulation of the random behavior of a single aggregation gating site. The concept of aggregation gating involves a voltage-dependent reversible conformational change and a voltage-independent reversible aggregation process. A site is assumed to consist of four hypothetical protein subunits. Based on these assumptions the model is defined by the scheme of transitions between 12 possible site configurations. The model can account for the phenomenon of charge immobilization in asymmetry current data of the voltage-clamped sodium conductance system. It predicts gating currents without a rising phase. A rising phase is obtained, however, if the model is subjected to conventional symmetrical pulse protocols for measuring asymmetry currents in the axon. Novel pulse protocols are given that do not result in a rising phase if applied to the aggregation model. Simplified transition schemes that describe the basic kinetic behavior of the potassium and the sodium conductance system are derived by eliminating transitions of negligible probability from the original scheme.     

Polymorphisms in genes of the steroid hormone biosynthesis and metabolism pathways and endometrial cancer risk

Objectives: The incidence of endometrial cancer has recently increased substantially and studies have shown that altered levels of exogenous and endogenous hormones are associated with individual variation in endometrial cancer risk. The environmental and reproductive risk factors that influence these hormones are well known, however, genetic variants involved in hormone biosynthesis and estrogen metabolism have not been well established in endometrial cancer. Methods: To determine whether polymorphisms in genes of the steroid hormone biosynthesis and metabolism pathways are associated with endometrial cancer risk, 28 polymorphisms in 18 genes were genotyped in 191 endometrial cancer cases and 291 healthy controls. Results: The GSTM1 deletion and the variant (GG) genotype of the CYP1B1 rs1800440 polymorphism were associated with a decreased risk of developing endometrial cancer. Furthermore, combinations of haplotypes in CYP1A1, CYP1B1 and GSTs were associated with a decreased risk. The analysis of the repeat polymorphisms revealed that women with the long repeat allele length of the ESR1 (GT)n repeat polymorphism were at an increased risk of developing endometrial cancer. Conversely, women with two long repeat length alleles of the (CAG)n repeat polymorphism in the AR correlated with a decrease in endometrial cancer risk compared to women with one or two alleles with the short repeat length. Conclusions: The findings are consistent with our hypothesis that variability in genes involved in steroidogenesis and estrogen metabolism may alter the risk of developing endometrial cancer, suggesting that they may be useful as biomarkers for genetic susceptibility to endometrial cancer.     

Motif-based searching in TOPS protein topology databases.

MOTIVATION: TOPS cartoons are a schematic ion of protein three-dimensional structures in two dimensions, and are used for understanding and manual comparison of protein folds. Recently, an algorithm that produces the cartoons automatically from protein structures has been devised and cartoons have been generated to represent all the structures in the structural databank. There is now a need to be able to define target topological patterns and to search the database for matching domains. RESULTS: We have devised a formal language for describing TOPS diagrams and patterns, and have designed an efficient algorithm to match a pattern to a set of diagrams. A pattern-matching system has been implemented, and tested on a database derived from all the current entries in the Protein Data Bank (15,000 domains). Users can search on patterns selected from a library of motifs or, alternatively, they can define their own search patterns. AVAILABILITY: The system is accessible over the Web at http://tops.ebi.ac.uk/tops