A HIF-1 target, ATIA, protects cells from apoptosis by modulating the mitochondrial thioredoxin, TRX2

The regulation of apoptosis is critical for controlling tissue homeostasis and preventing tumor formation and growth. Reactive oxygen species (ROS) generation plays a key role in such regulation. Here, we describe a HIF-1 target, Vasn/ATIA (anti-TNFα-induced apoptosis), which protects cells against TNFα- and hypoxia-induced apoptosis. Through the generation of ATIA knockout mice, we show that ATIA protects cells from apoptosis through regulating the function of the mitochondrial antioxidant, thioredoxin-2, and ROS generation. ATIA is highly expressed in human glioblastoma, and ATIA knockdown in glioblastoma cells renders them sensitive to hypoxia-induced apoptosis. Therefore, ATIA is not only a HIF-1 target that regulates mitochondrial redox pathways but also a potentially diagnostic marker and therapeutic target in human glioblastoma.     

Accuracy of side-chain prediction upon near-native protein backbones generated by ab initio folding methods

The ab initio folding problem can be divided into two sequential tasks of approximately equal computational complexity: the generation of native-like backbone folds and the positioning of side chains upon these backbones. The prediction of side-chain conformation in this context is challenging, because at best only the near-native global fold of the protein is known. To test the effect of displacements in the protein backbones on side-chain prediction for folds generated ab initio, sets of near-native backbones (≤ 4 Å Cα RMS error) for four small proteins were generated by two methods. The steric environment surrounding each residue was probed by placing the side chains in the native conformation on each of these decoys, followed by torsion-space optimization to remove steric clashes on a rigid backbone. We observe that on average 40% of the χ1 angles were displaced by 40° or more, effectively setting the limits in accuracy for side-chain modeling under these conditions. Three different algorithms were subsequently used for prediction of side-chain conformation. The average prediction accuracy for the three methods was remarkably similar: 49% to 51% of the χ1 angles were predicted correctly overall (33% to 36% of the χ1+2 angles). Interestingly, when the inter-side-chain interactions were disregarded, the mean accuracy increased. A consensus approach is described, in which side-chain conformations are defined based on the most frequently predicted χ angles for a given method upon each set of near-native backbones. We find that consensus modeling, which de facto includes backbone flexibility, improves side-chain prediction: χ1 accuracy improved to 51–54% (36–42% of χ1+2). Implications of a consensus method for ab initio protein structure prediction are discussed. Proteins 33:204–217, 1998. © 1998 Wiley-Liss, Inc.     

Isolation and partial characterization of endothelial cell extracellular complexes

Human endothelial cells release components into the growth medium that stimulate cell-substratum adhesion. Several macromolecular components were isolated by ultracentrifugation of the endothelial cell conditioned medium. The components were heterogeneous, consisting of several sizes when examined by sedimentation velocity and gel filtration. When the extracellular components were evaluated by electron microscopy, structurally discrete particles were observed. The extracellular components and the complexes mediated cell-substratum adhesion to both human umbilical and arterial endothelial cells. The majority of the extracellular components that promote endothelial cell adhesion were pelleted by ultracentrifugation. Although the complexes contained fibronectin, antibodies to fibronectin did not inhibit cell adhesion to the complexes. Significant inhibition of endothelial cell adhesion was observed in the presence of heparin and heparan sulfate. The supernatant fraction following ultracentrifugation of the growth medium contained a component that suppressed endothelial cell adhesion to culture dishes coated with fibronectin, type I collagen, and endothelial cell complexes. SDS-polyacrylamide gel electrophoresis indicated that the complexes contained several components, and the majority of the large-molecular-weight components were pelleted by ultracentrifugation. The conditioned medium from human endothelial cells contains specific complexes that promote cell-substratum adhesion and components that suppress cell-substratum adhesion.     

Waist Circumference,BMI, and Visceral Adipose Tissue in White Women and Women of African Descent

Although waist circumference (WC) is a marker of visceral adipose tissue (VAT), WC cut‐points are based on BMI category. We compared WC‐BMI and WC‐VAT relationships in blacks and whites. Combining data from five studies, BMI and WC were measured in 1,409 premenopausal women (148 white South Africans, 607 African‐Americans, 186 black South Africans, 445 West Africans, 23 black Africans living in United States). In three of five studies, participants had VAT measured by computerized tomography (n = 456). Compared to whites, blacks had higher BMI (29.6 ± 7.6 (mean ± s.d.) vs. 27.6 ± 6.6 kg/m², P = 0.001), similar WC (92 ± 16 vs. 90 ± 15 cm, P = 0.27) and lower VAT (64 ± 42 vs. 101 ± 59 cm², P < 0.001). The WC‐BMI relationship did not differ by race (blacks: β (s.e.) WC = 0.42 (.01), whites: β (s.e.) WC = 0.40 (0.01), P = 0.73). The WC‐VAT relationship was different in blacks and whites (blacks: β (s.e.) WC = 1.38 (0.11), whites: β (s.e.) WC = 3.18 (0.21), P < 0.001). Whites had a greater increase in VAT per unit increase in WC. WC‐BMI and WC‐VAT relationships did not differ among black populations. As WC‐BMI relationship did not differ by race, the same BMI‐based WC guidelines may be appropriate for black and white women. However, if WC is defined by VAT, race‐specific WC thresholds are required.     

Architectural design influences the diversity and structure of the built environment microbiome

Buildings are complex ecosystems that house trillions of microorganisms interacting with each other, with humans and with their environment. Understanding the ecological and evolutionary processes that determine the diversity and composition of the built environment microbiome—the community of microorganisms that live indoors—is important for understanding the relationship between building design, biodiversity and human health. In this study, we used high-throughput sequencing of the bacterial 16S rRNA gene to quantify relationships between building attributes and airborne bacterial communities at a health-care facility. We quantified airborne bacterial community structure and environmental conditions in patient rooms exposed to mechanical or window ventilation and in outdoor air. The phylogenetic diversity of airborne bacterial communities was lower indoors than outdoors, and mechanically ventilated rooms contained less diverse microbial communities than did window-ventilated rooms. Bacterial communities in indoor environments contained many taxa that are absent or rare outdoors, including taxa closely related to potential human pathogens. Building attributes, specifically the source of ventilation air, airflow rates, relative humidity and temperature, were correlated with the diversity and composition of indoor bacterial communities. The relative abundance of bacteria closely related to human pathogens was higher indoors than outdoors, and higher in rooms with lower airflow rates and lower relative humidity. The observed relationship between building design and airborne bacterial diversity suggests that we can manage indoor environments, altering through building design and operation the community of microbial species that potentially colonize the human microbiome during our time indoors.     

The status and conservation of the Cape Gannet Morus capensis

The Cape Gannet Morus capensis is one of several seabird species endemic to the Benguela upwelling ecosystem (BUS) but whose population has recently decreased, leading to an unfavourable IUCN Red List assessment. Application of ‘JARA’ (‘Just Another Red-List Assessment,’ a Bayesian state-space tool used for IUCN Red List assessments) to updated information on the areas occupied by Cape Gannets and the nest densities of breeding birds at their six colonies, suggested that the species should be classified as Vulnerable. However, the rate of decrease of Cape Gannets in their most-recent generation exceeded that of the previous generation, primarily as a result of large decreases at Bird Island, Lambert’s Bay, and Malgas Island, off South Africa’s west coast (the western part of their range). Since the 1960s, there has been an ongoing redistribution of the species from northwest to southeast around southern Africa, and ～70% of the population now occurs on the south coast of South Africa, at Bird Island in Algoa Bay, on the eastern border of the BUS. Recruitment rather than adult survival may be limiting the present population; however, information on the seabird’s demographic parameters and mortality in fisheries is lacking for colonies in the northern part of the BUS. Presently, major threats to Cape Gannet include: substantially decreased availability of their preferred prey in the west; heavy mortalities of eggs, chicks and fledglings at and around colonies, inflicted by Cape Fur Seals Arctocephalus pusillus and other seabirds; substantial disturbance at colonies caused by Cape Fur Seals attacking adult gannets ashore; oiling; and disease.     

Membrane localization diversity of TPK channels and their physiological role

Potassium (K) is one of the major nutrients that is essential for plant growth and development. The majority of cellular K⁺ resides in the vacuole and tonoplast K⁺ channels of the TPK (Two Pore K) family are main players in cellular K⁺ homeostasis. All TPK channels were previously reported to be expressed in the tonoplast of the large central lytic vacuole (LV) except for one isoform in Arabidopsis that resides in the plasma membrane. However, plant cells often contain more than one type of vacuole that coexist in the same cell. We recently showed that two TPK isoforms (OsTPKa and OsTPKb) from Oryza sativa localize to different vacuoles with OsTPKa predominantly found in the LV tonoplast and OsTPKb primarily in smaller compartments that resemble small vacuoles (SVs). Our study further revealed that it is the C-terminal domain that determines differential targeting of OsTPKa and OsTPKb. Three C-terminal amino acids were particularly relevant for targeting TPKs to their respective endomembranes. In this addendum we further evaluate how the different localization of TPKa and TPKb impact on their physiological role and how TPKs provide a potential tool to study the physiology of different types of vacuole.Key words: TPK channels, small vacuoles, vacuolar targeting, potassiumThe roles of plant vacuolar K⁺ channels are diverse and include potassium homeostasis, turgor regulation and responses to abiotic stress. Vacuolar K⁺-selective channels belong to two-pore K⁺ (TPK) channel families which have been found in genomes of many plant species such as Arabidopsis, poplar, Physcomitrella, Eucalyptus, barley, potato, rice and tobacco (Fig. 1). TPKs have structural similarity to mammalian “tandem P domain” channels with a secondary structure that contains four transmembrane domains and two pore regions (Fig. 2).^1–5 TPK channels have pore regions with a GYGD signature that endows K⁺ selectivity and a variable number of Ca²⁺ binding EF domains in the C terminus.^3–8 One of the best characterized members of the TPK family is AtTPK1 from Arabidopsis thaliana. AtTPK1 activity is voltage independent but sensitive to cytosolic Ca²⁺, cytosolic pH and N-terminal phosphorylation by 14-3-3 proteins.^5,6,8,9 In Arabidopsis, AtTPK1 expresses in the large lytic vacuole (LV) and plays roles in cellular K⁺ homeostasis, K⁺-release during stomatal closure and seed germination.^4,5 Other members of the Arabidopsis TPK family (AtTPK2, AtTPK3, AtTPK5) have been shown to localize to the LV but also showed some expression in smaller, vesicle-like, compartments.⁴ However, none of these isoforms appears to form functional channels in planta although our experiments with heterologous expression of AtTPK3 and AtTPK5 in the K⁺ uptake deficient E. coli LB2003 demonstrates complementation of bacterial growth phenotype (Isayenkov S, et al. unpublished results). Equally intriguing, is the plasma membrane localization of the Arabidopsis TPK4 isoform, in spite of its sequence being very similar to that of other TPKs.¹⁰Open in a separate windowFigure 1Phylogenetic tree of plant TPKs. The three main clusters of TPKs comprise: Cluster 1 with AtTPK1-like channels; Cluster 2 with AtTPK3/TPK5-like channels; Cluster 3 with barley HvTPKb. Bootstrap analysis was performed using ‘Molecular Evolutionary Genetics Analysis, MEGA4’ software available at www.megasoftware.net/mega4/megaOpen in a separate windowFigure 2Two-pore potassium channel secondary structure. TPK channels comprise four transmembrane domains (1–4) and two pore regions (P) per subunit. Functional channels are formed from two subunits. In most TPKs, both P regions contain a K⁺ selectivity signature, GYGD. However, the tobacco NtTPKa isoform has different motifs in the second P domain. In the N terminal region, TPKs have a 14-3-3 binding domain that impact on channel activity, with the binding of 14-3-3 protein leading to channel activation. C-termini of TPKs show a varying number of putative Ca²⁺ binding “EF hands” which may vary from zero to two.     

Exploring the Role of a Conserved Class A Residue in the {Omega}-Loop of KPC-2 β-Lactamase: A MECHANISM FOR CEFTAZIDIME HYDROLYSIS

Gram-negative bacteria harboring KPC-2, a class A β-lactamase, are resistant to all β-lactam antibiotics and pose a major public health threat. Arg-164 is a conserved residue in all class A β-lactamases and is located in the solvent-exposed Ω-loop of KPC-2. To probe the role of this amino acid in KPC-2, we performed site-saturation mutagenesis. When compared with wild type, 11 of 19 variants at position Arg-164 in KPC-2 conferred increased resistance to the oxyimino-cephalosporin, ceftazidime (minimum inhibitory concentration; 32→128 mg/liter) when expressed in Escherichia coli. Using the R164S variant of KPC-2 as a representative β-lactamase for more detailed analysis, we observed only a modest 25% increase in k(cat)/K(m) for ceftazidime (0.015→0.019 μm(-1) s(-1)). Employing pre-steady-state kinetics and mass spectrometry, we determined that acylation is rate-limiting for ceftazidime hydrolysis by KPC-2, whereas deacylation is rate-limiting in the R164S variant, leading to accumulation of acyl-enzyme at steady-state. CD spectroscopy revealed that a conformational change occurred in the turnover of ceftazidime by KPC-2, but not the R164S variant, providing evidence for a different form of the enzyme at steady state. Molecular models constructed to explain these findings suggest that ceftazidime adopts a unique conformation, despite preservation of Ω-loop structure. We propose that the R164S substitution in KPC-2 enhances ceftazidime resistance by proceeding through "covalent trapping" of the substrate by a deacylation impaired enzyme with a lower K(m). Future antibiotic design must consider the distinctive behavior of the Ω-loop of KPC-2.     

Elevated levels of fibrinogen-derived endogenous citrullinated peptides in synovial fluid of rheumatoid arthritis patients

Introduction

Rheumatoid arthritis (RA) is an autoimmune disease characterized by inflammation of the joints and the presence of autoantibodies directed against proteins containing the non-standard arginine-derived amino acid citrulline. The protein fibrinogen, which has an essential role in blood clotting, is one of the most prominent citrullinated autoantigens in RA, particularly because it can be found in the inflamed tissue of affected joints. Here, we set out to analyze the presence of citrullinated endogenous peptides in the synovial fluid of RA and arthritic control patients.

Methods

Endogenous peptides were isolated from the synovial fluid of RA patients and controls by filtration and solid phase extraction. The peptides were identified and quantified using high-resolution liquid chromatography-mass spectrometry.

Results

Our data reveal that the synovial fluid of RA patients contains soluble endogenous peptides, derived from fibrinogen, containing significant amounts of citrulline residues and, in some cases, also phosphorylated serine. Several citrullinated peptides are found to be more abundantly present in the synovial fluid of RA patients compared to patients suffering from other inflammatory diseases affecting the joints.

Conclusions

The increased presence of citrullinated peptides in RA patients points toward a possible specific role of these peptides in the immune response at the basis of the recognition of citrullinated peptides and proteins by RA patient autoantibodies.