The accuracy of several multiple sequence alignment programs for proteins

Background  

There have been many algorithms and software programs implemented for the inference of multiple sequence alignments of protein and DNA sequences. The "true" alignment is usually unknown due to the incomplete knowledge of the evolutionary history of the sequences, making it difficult to gauge the relative accuracy of the programs.     

On single and multiple models of protein families for the detection of remote sequence relationships

Background  

The detection of relationships between a protein sequence of unknown function and a sequence whose function has been characterised enables the transfer of functional annotation. However in many cases these relationships can not be identified easily from direct comparison of the two sequences. Methods which compare sequence profiles have been shown to improve the detection of these remote sequence relationships. However, the best method for building a profile of a known set of sequences has not been established. Here we examine how the type of profile built affects its performance, both in detecting remote homologs and in the resulting alignment accuracy. In particular, we consider whether it is better to model a protein superfamily using a single structure-based alignment that is representative of all known cases of the superfamily, or to use multiple sequence-based profiles each representing an individual member of the superfamily.     

A novel surface protein of Trichomonas vaginalis is regulated independently by low iron and contact with vaginal epithelial cells

Background  

Trichomonosis caused by Trichomonas vaginalis is the number one, non-viral sexually transmitted disease (STD) that affects more than 250 million people worldwide. Immunoglobulin A (IgA) has been implicated in resistance to mucosal infections by pathogens. No reports are available of IgA-reactive proteins and the role, if any, of this class of antibody in the control of this STD. The availability of an IgA monoclonal antibody (mAb) immunoreactive to trichomonads by whole cell (WC)-ELISA prompted us to characterize the IgA-reactive protein of T. vaginalis.     

Decarboxylation is a significant reaction pathway for photolabile calcium chelators and related compounds.

Photolysis of amino acids that bear a 2-nitrobenzyl protecting group on the amino nitrogen involves a normal 2-nitrobenzyl-type photocleavage to release the amino acid but also a second mechanistic pathway, that appears to be initiated by single electron transfer from the amino group to the excited state of the nitroaromatic. The end result of this pathway is photodecarboxylation. Quantitative experiments suggest that this latter pathway can contribute between 10 and 80% of the total reaction flux in different compounds. It appears that the aminium radical, the first product of the single electron transfer, can be intercepted by certain amino acids, resulting in a transfer of decarboxylation to this "sacrificial" amino acid. Possible implications for precise details of calcium release from photolabile derivatives of EDTA and EGTA are discussed.     

Motion and twisting of magnetic particles ingested by alveolar macrophages in the human lung: effect of smoking and disease

Background

Magnetic microparticles being ingested by alveolar macrophages can be used as a monitor for intracellular phagosome motions and cytoskeletal mechanical properties. These studies can be performed in the human lung after voluntary inhalation. The influence of cigarette smoking and lung diseases on cytoskeleton dependent functions was studied.

Methods

Spherical 1.3 μm diameter ferrimagnetic iron oxide particles were inhaled by 17 healthy volunteers (40 – 65 years), 15 patients with sarcoidosis (SAR), 12 patients with idiopathic pulmonary fibrosis (IPF), and 18 patients with chronic obstructive bronchitis (COB). The retained particles were magnetized and aligned in an external 100 mT magnetic field. All magnetized particles induce a weak magnetic field of the lung, which was detected by a sensitive SQUID (superconducting quantum interference device) sensor. Cytoskeletal reorganizations within macrophages and intracellular transport cause stochastic magnetic dipole rotations, which are reflected in a decay of the magnetic lung field, called relaxation. Directed phagosome motion was induced in a weak magnetic twisting field. The resistance of the cytoplasm to particle twisting was characterized by the viscosity and the stiffness (ratio between stress to strain) of the cytoskeleton.

Results

One week after particle inhalation and later macrophage motility (relaxation) and cytoskeletal stiffness was not influenced by cigarette smoking, neither in healthy subjects, nor in the patients. Patients with IPF showed in tendency a faster relaxation (p = 0.06). Particle twisting revealed a non-Newtonian viscosity with a pure viscous and a viscoelastic compartment. The viscous shear was dominant, and only 27% of the shear recoiled and reflected viscoelastic properties. In patients with IPF, the stiffness was reduced by 60% (p < 0.02). An analysis of the shear rate and stress dependence of particle twisting allows correlating the rheological compartments to cytoskeletal subunits, in which microtubules mediate the pure viscous (non-recoverable) shear and microfilaments mediate the viscoelastic (recoverable) behavior. The missing correlation between relaxation and particle twisting shows that both stochastic and directed phagosome motion reflect different cytoskeletal mechanisms.

Conclusion

Faster relaxation and a soft cytoskeleton in patients with IPF indicate alterations in cytoskeleton dependent functions of alveolar macrophages, which may cause dysfunction's in the alveolar defense, like a slower migration, a retarded phagocytosis, a disturbed phagosome lysosome fusion and an impaired clearance.

     

The adhering junctions of valvular interstitial cells: molecular composition in fetal and adult hearts and the comings and goings of plakophilin-2 in situ, in cell culture and upon re-association with scaffolds

The interstitial cells of cardiac valves represent one of the most frequent cell types in the mammalian heart. In order to provide a cell and molecular biological basis for the growth of isolated valvular interstitial cells (VICs) in cell culture and for the use in re-implantation surgery we have examined VICs in situ and in culture, in fetal, postnatal and adult hearts, in re-associations with scaffolds of extracellular matrix (ECM) material and decellularized heart valves. In all four mammalian species examined (human, bovine, porcine and ovine), the typical mesenchymal-type cell-cell adherens junctions (AJs) connecting VICs appear as normal N-cadherin based puncta adhaerentia. Their molecular ensemble, however, changes under various growth conditions insofar as plakophilin-2 (Pkp2), known as a major cytoplasmic plaque component of epithelial desmosomes, is recruited to and integrated in the plaques of VIC-AJs as a major component under growth conditions characterized by enhanced proliferation, i.e., in fetal heart valves and in cell cultures. Upon re-seeding onto decellularized heart valves or in stages of growth in association with artificial scaffolds, Pkp2 is - for the most part - lost from the AJs. As Pkp2 has recently also been detected in AJs of cardiac myxomata and diverse other mesenchymal tumors, the demonstrated return to the normal Pkp2-negative state upon re-association with ECM scaffolds and decellularized heart valves may now provide a safe basis for the use of cultured VICs in valve replacement surgery. Even more surprising, this type of transient acquisition of Pkp2 has also been observed in distinct groups of endothelial cells of the endocardium, where it seems to correspond to the cell type ready for endothelial-mesenchymal transition (EMT).     

In vivo imaging of immunotoxin treatment using Katushka-transfected A-431 cells in a murine xenograft tumour model

Purpose

Preclinical in vivo analyses of treatment responses are an important prerequisite to evaluate new therapeutics. Molecular in vivo imaging in the far red (FR)/near infra red (NIR) is a promising method, as it enables measurements at different time points in individual animals, thereby reducing the number of animals required, while increasing statistical significance. Here, we show the establishment of a method to monitor response to treatment using fluorescent cells, expressing the epidermal growth factor receptor (EGFR), a target already used in therapy.

Methods

We transfected A-431 tumour cells with the far red–emitting protein Katushka (Kat2), resulting in strong fluorescence allowing for the monitoring of tumour growth when implanted in BALB/c nu/nu mice with a CRi Maestro in vivo imager. We targeted A-431 cells with a previously reported immunotoxin (IT), consisting of the anti-EGFR antibody single-chain variable fragment (scFv) 425, fused to Pseudomonas aeruginosa Exotoxin A’ (ETA’). In addition, EGFR expression was verified using the 425(scFv) conjugated to a NIR dye BG-747 through a SNAP-tag linker.

Results

The results show the feasibility to evaluate response to treatment in vivo by FR imaging, while at the same location detecting EGFR expression. Treatment with 425(scFv)-ETA’ resulted in decelerated tumour growth, while not affecting the overall health of the animals. This is in contrast to treatment with Doxorubicin, which, although decreasing the tumour size, resulted in poor health.

Conclusions

We developed a novel method to non-invasively determine treatment responses by in vivo imaging of multiple parameters which showed the efficacy of 425(scFv)-ETA’.     

Metabolically induced heteroplasmy shifting and l-arginine treatment reduce the energetic defect in a neuronal-like model of MELAS

The m.3243A>G variant in the mitochondrial tRNA(Leu(UUR)) gene is a common mitochondrial DNA (mtDNA) mutation. Phenotypic manifestations depend mainly on the heteroplasmy, i.e. the ratio of mutant to normal mtDNA copies. A high percentage of mutant mtDNA is associated with a severe, life-threatening neurological syndrome known as MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes). MELAS is described as a neurovascular disorder primarily affecting the brain and blood vessels, but the pathophysiology of the disease is poorly understood. We developed a series of cybrid cell lines at two different mutant loads: 70% and 100% in the nuclear background of a neuroblastoma cell line (SH-SY5Y). We investigated the impact of the mutation on the metabolism and mitochondrial respiratory chain activity of the cybrids. The m.3243A>G mitochondrial mutation induced a metabolic switch towards glycolysis in the neuronal cells and produced severe defects in respiratory chain assembly and activity. We used two strategies to compensate for the biochemical defects in the mutant cells: one consisted of lowering the glucose content in the culture medium, and the other involved the addition of l-arginine. The reduction of glucose significantly shifted the 100% mutant cells towards the wild-type, reaching a 90% mutant level and restoring respiratory chain complex assembly. The addition of l-arginine, a nitric oxide (NO) donor, improved complex I activity in the mutant cells in which the defective NO metabolism had led to a relative shortage of NO. Thus, metabolically induced heteroplasmy shifting and l-arginine therapy may constitute promising therapeutic strategies against MELAS.     

Weak interactions govern the viscosity of concentrated antibody solutions: high-throughput analysis using the diffusion interaction parameter

Weak protein-protein interactions are thought to modulate the viscoelastic properties of concentrated antibody solutions. Predicting the viscoelastic behavior of concentrated antibodies from their dilute solution behavior is of significant interest and remains a challenge. Here, we show that the diffusion interaction parameter (k(D)), a component of the osmotic second virial coefficient (B(2)) that is amenable to high-throughput measurement in dilute solutions, correlates well with the viscosity of concentrated monoclonal antibody (mAb) solutions. We measured the k(D) of 29 different mAbs (IgG(1) and IgG(4)) in four different solvent conditions (low and high ion normality) and found a linear dependence between k(D) and the exponential coefficient that describes the viscosity concentration profiles (|R| ≥ 0.9). Through experimentally measured effective charge measurements, under low ion normality where the electroviscous effect can dominate, we show that the mAb solution viscosity is poorly correlated with the mAb net charge (|R| ≤ 0.6). With this large data set, our results provide compelling evidence in support of weak intermolecular interactions, in contrast to the notion that the electroviscous effect is important in governing the viscoelastic behavior of concentrated mAb solutions. Our approach is particularly applicable as a screening tool for selecting mAbs with desirable viscosity properties early during lead candidate selection.     

Genetic structure in a dynamic baboon hybrid zone corroborates behavioural observations in a hybrid population

Behaviour and genetic structure are intimately related: mating patterns and patterns of movement between groups or populations influence the movement of genetic variation across the landscape and from one generation to the next. In hybrid zones, the behaviour of the hybridizing taxa can also impact the incidence and outcome of hybridization events. Hybridization between yellow baboons and anubis baboons has been well documented in the Amboseli basin of Kenya, where more anubis-like individuals tend to experience maturational and reproductive advantages. However, it is unknown whether these advantages are reflected in the genetic structure of populations surrounding this area. Here, we used microsatellite genotype data to evaluate the structure and composition of baboon populations in southern Kenya. Our results indicate that, unlike for mitochondrial DNA, microsatellite-based measures of genetic structure concord with phenotypically based taxonomic distinctions and that the currently active hybrid zone is relatively narrow. Isolation with migration analysis revealed asymmetric gene flow in this region from anubis populations into yellow populations, in support of the anubis-biased phenotypic advantages observed in Amboseli. Populations that are primarily yellow but that receive anubis gene flow exhibit higher levels of genetic diversity than yellow populations far from the introgression front. Our results support previous work that indicates a long history of hybridization and introgression among East African baboons. Specifically, it suggests that anubis baboons are in the process of gradual range expansion into the range of yellow baboons, a pattern potentially explained by behavioural and life history advantages that correlate with anubis ancestry.