Characterization of the proteins of bacterial strain isolated from contaminated site involved in heavy metal resistance--a proteomic approach

The present study describes response of a bacterial strain isolated from a polluted river to heavy metal toxicity. The bacterium was identified to be Klebsiella pneumoniae by biochemical tests using API 20E strips and 16S ribotyping. The isolate was studied for its tolerance to two heavy metals, i.e., cobalt (Co(2+)) and lead (Pb(2+)) by growing it in citrate mineral medium (CMM). Proteomic approach involving two-dimensional polyacrylamide gel electrophoresis (2D PAGE) and mass spectrometry (MS) was used to identify the differentially expressed proteins under heavy metal stress. Two of the differentially expressed proteins were identified to be l-isoaspartate protein carboxymethyltransferase type II and DNA gyrase A. To our knowledge, this is for the first time that K. pneumoniae has been reported to be present in metal contaminated site and l-isoaspartate protein carboxymethyltransferase type II protein to be over expressed under heavy metal stress. The role of these proteins in metal tolerance is discussed.     

Impact of a Glaucoma Severity Index on Results of Trabectome Surgery: Larger Pressure Reduction in More Severe Glaucoma

Purpose

To stratify outcomes of trabectome-mediated ab interno trabeculectomy (AIT) by glaucoma severity using a simple and clinically useful glaucoma index. Based on prior data of trabectome after failed trabeculectomy, we hypothesized that more severe glaucoma might have a relatively more reduced facility compared to mild glaucoma and respond with a larger IOP reduction to trabecular meshwork ablation.

Methods

Patients with primary open angle glaucoma who had undergone AIT without any other same session surgery and without any second eye surgery during the following 12 months were analyzed. Eyes of patients that had less than 12 months follow up or were diagnosed with neovascular glaucoma were excluded. A glaucoma index (GI) was created to capture glaucoma severity based on visual field, number of preoperative medications, and preoperative IOP. Visual field (VF) was separated into 3 categories: mild, moderate, and advanced (assigned 1, 2, and 3 points, respectively). Preoperative number of medications (meds) was divided into 4 categories: ≤1, 2, 3 or ≥4, and assigned with a value of 1 to 4. Baseline IOP (IOP) was divided into 3 categories: <20 mmHg, 20–29 mmHg, and greater than 30 mmHg and assigned with 1 to 3 points. GI was defined as IOP × meds × VF and separated into 4 groups: <6 (Group 1), 6–12 (Group 2), >12–18 (Group 3) and >18 (Group 4). Linear regression was used to determine if there was an association between GI group and IOP reduction after one year or age, gender, race, diagnosis, cup to disc (C/D) ratio, and Shaffer grade.

Results

Out of 1340 patients, 843 were included in the analysis. The GI group distribution was GI1 = 164, GI2 = 202, GI3 = 260, and GI4 = 216. Mean IOP reduction after one year was 4.0±5.4, 6.4±5.8, 9.0±7.6, 12.0±8.0 mmHg for GI groups 1 to 4, respectively. Linear regression showed that IOP reduction was associated with GI group after adjusting for age, gender, race, diagnosis, cup to disc ratio, and Shaffer grade. Each GI group increase of 1 was associated with incremental IOP reductions of 2.95±0.29 mmHg. Success rate at 12 months was 90%, 77%, 77%, and 71% for GI groups 1 to 4. The log-rank test suggested significant differences between GI groups.

Conclusion

A simple glaucoma index, GI, was created to capture glaucoma severity and a relative resistance to treatment. A higher GI was associated with a larger IOP reduction in trabectome surgery. This indicates that there is a role for AIT beyond mild glaucoma and ocular hypertension.     

Role for the alpha-helix in aberrant protein aggregation

Is the alpha-helix structure capable of triggering the formation of aberrant protein aggregates? To answer this question, we investigate the in vitro aggregation of tau protein in the presence of the helix-inducing agent TFE. Tau is a natively unfolded protein that binds to microtubules and forms aggregates in Alzheimer's disease. We find that full-length tau has residual alpha-helix structure, which is further enhanced by three mutations involved in genetic neurological disorders. TFE concentrations matching an alpha-helical content of 40% in full-length tau and the triple mutant induce the formation of aggregates that are morphologically and structurally heterogeneous. A simple dilution experiment reveals that heterogeneity results from the competition between alpha-helical fibrillar aggregates and more classical amyloid-like aggregates. The alpha-helical aggregates are more resilient to dilution and have the spectroscopic features of alpha-helical coiled coils. We propose a general mechanism by which intrinsically stable alpha-helices can associate into aggregates with only coarse coiled-coil symmetry. In tau, high intrinsic alpha-helix stability and coarse coiled-coil symmetry could be byproducts of its biological function.     

MSH1-Induced Non-Genetic Variation Provides a Source of Phenotypic Diversity in Sorghum bicolor

MutS Homolog 1 (MSH1) encodes a plant-specific protein that functions in mitochondria and chloroplasts. We showed previously that disruption or suppression of the MSH1 gene results in a process of developmental reprogramming that is heritable and non-genetic in subsequent generations. In Arabidopsis, this developmental reprogramming process is accompanied by striking changes in gene expression of organellar and stress response genes. This developmentally reprogrammed state, when used in crossing, results in a range of variation for plant growth potential. Here we investigate the implications of MSH1 modulation in a crop species. We found that MSH1-mediated phenotypic variation in Sorghum bicolor is heritable and potentially valuable for crop breeding. We observed phenotypic variation for grain yield, plant height, flowering time, panicle architecture, and above-ground biomass. Focusing on grain yield and plant height, we found some lines that appeared to respond to selection. Based on amenability of this system to implementation in a range of crops, and the scope of phenotypic variation that is derived, our results suggest that MSH1 suppression provides a novel approach for breeding in crops.     

Heart rate measures in blind cave crayfish during environmental disturbances and social interactions

Most animals continually assess the environment in which they live and alter their behavior according to various stimuli. As an observer, one looks for changes in a behavior indicating that an animal responded to a particular event. When the animal does not make significant behavioral changes as measured by bodily movements, the animal may be characterized as unresponsive to a given stimulus. This study demonstrates that when behavioral body movements can not be observed an internal physiological measure of heart rate (HR) shows dramatic changes following presentation of defined stimuli. This study used the blind cave crayfish and examined their responsiveness to the following stimuli: light (infrared, dim red, and white), water-borne vibrations, removal of water, olfactory cues, and social interaction with partners. This study demonstrates that there is substantial individual variation of HR at basal levels and with the intensity of an social interaction. We find HR is a reasonable measure of the responsiveness of blind cave crayfish to given stimuli even in the absence of observable behavioral changes. This enables the observer to determine if an individual is responsive to and making an assessment of particular cues.     

Efficient and repetitive production of hematopoietic and endothelial cells from feeder-free monolayer culture system of primate embryonic stem cells

We have established an innovative culture system for the efficient differentiation of hematopoietic and endothelial cells from primate embryonic stem (ES) cells without feeder cells, embryoid bodies, or cell-sorting processes. After several days' culture in murine stromal OP9-conditioned medium supplemented with a cytokine cocktail on collagen-coated dishes, ES cells differentiated into a very unique population of cells with a finger-like appearance. These finger-like cells were positive for mesodermal and/or hemangioblastic markers of kinase insert domain receptor (KDR) and T-cell acute lymphocytic leukemia 1 (TAL1), and produced large amounts of protein tyrosine phosphatase, receptor type, C-positive hematopoietic cells. These hematopoietic cells showed the morphology of immature hematopoietic cells, formed blast cell colonies with high efficiency, and were positive for CD34 antigen, KDR, TAL1, and GATA binding protein 1, suggesting that these blast cells are equivalent to the multipotent hematopoietic progenitor cells. Moreover, they produced functional macrophages in murine stromal MS-5-conditioned medium and primitive erythroblasts in the presence of erythropoietin. The finger-like cells, putative mesodermal progenitors and/or hemangioblasts, actively proliferated and repetitively produced hematopoietic cells as long as they were maintained on the original dish. By contrast, the majority of the finger-like cells differentiated into endothelial cells with specific markers and specific functions after transfer to fresh dishes, indicating that conditions established in the original dish supported the proliferation and hematopoietic differentiation of the finger-like cells. Our method provides a highly controllable culture protocol for repetitive production of hematopoietic and endothelial cells from feeder-free monolayer cultivation of primate ES cells.     

A simple method for estimating global DNA methylation using bisulfite PCR of repetitive DNA elements

We report a method for studying global DNA methylation based on using bisulfite treatment of DNA and simultaneous PCR of multiple DNA repetitive elements, such as Alu elements and long interspersed nucleotide elements (LINE). The PCR product, which represents a pool of approximately 15000 genomic loci, could be used for direct sequencing, selective restriction digestion or pyrosequencing, in order to quantitate DNA methylation. By restriction digestion or pyrosequencing, the assay was reproducible with a standard deviation of only 2% between assays. Using this method we found that almost two-thirds of the CpG methylation sites in Alu elements are mutated, but of the remaining methylation target sites, 87% were methylated. Due to the heavy methylation of repetitive elements, this assay was especially useful in detecting decreases in DNA methylation, and this assay was validated by examining cell lines treated with the methylation inhibitor 5-aza-2′deoxycytidine (DAC), where we found a 1–16% decrease in Alu element and 18–60% LINE methylation within 3 days of treatment. This method can be used as a surrogate marker of genome-wide methylation changes. In addition, it is less labor intensive and requires less DNA than previous methods of assessing global DNA methylation.     

Molecular Disruption of the Power Stroke in the ATP-binding Cassette Transport Protein MsbA

ATP-binding cassette transporters affect drug pharmacokinetics and are associated with inherited human diseases and impaired chemotherapeutic treatment of cancers and microbial infections. Current alternating access models for ATP-binding cassette exporter activity suggest that ATP binding at the two cytosolic nucleotide-binding domains provides a power stroke for the conformational switch of the two membrane domains from the inward-facing conformation to the outward-facing conformation. In outward-facing crystal structures of the bacterial homodimeric ATP-binding cassette transporters MsbA from Gram-negative bacteria and Sav1866 from Staphylococcus aureus, two transmembrane helices (3 and 4) in the membrane domains have their cytoplasmic extensions in close proximity, forming a tetrahelix bundle interface. In biochemical experiments on MsbA from Escherichia coli, we show for the first time that a robust network of inter-monomer interactions in the tetrahelix bundle is crucial for the transmission of nucleotide-dependent conformational changes to the extracellular side of the membrane domains. Our observations are the first to suggest that modulation of tetrahelix bundle interactions in ATP-binding cassette exporters might offer a potent strategy to alter their transport activity.