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.     

TNFα and IL-1β are mediated by both TLR4 and Nod1 pathways in the cultured HAPI cells stimulated by LPS

A growing body of evidence recently suggests that glial cell activation plays an important role in several neurodegenerative diseases and neuropathic pain. Microglia in the central nervous system express toll-like receptor 4 (TLR4) that is traditionally accepted as the primary receptor of lipopolysaccharide (LPS). LPS activates TLR4 signaling pathways to induce the production of proinflammatory molecules. In the present studies, we verified the LPS signaling pathways using cultured highly aggressively proliferating immortalized (HAPI) microglial cells. We found that HAPI cells treated with LPS upregulated the expression of TLR4, phospho-JNK (pJNK) and phospho-NF-κB (pNF-κB), TNFα and IL-1β. Silencing TLR4 with siRNA reduced the expression of pJNK, TNFα and IL-1β, but not pNF-κB in the cells. Inhibition of JNK with SP600125 (a JNK inhibitor) decreased the expression of TNFα and IL-1β. Unexpectedly, we found that inhibition of Nod1 with ML130 significantly reduced the expression of pNF-κB. Inhibition of NF-κB also reduced the expression of TNFα and IL-1β. Nod1 ligand, DAP induced the upregulation of pNF-κB which was blocked by Nod1 inhibitor. These data indicate that LPS-induced pJNK is TLR4-dependent, and that pNF-κB is Nod1-dependent in HAPI cells treated with LPS. Either TLR4-JNK or Nod1-NF-κB pathways is involved in the expression of TNFα and IL-1β.     

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.     

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.     

Incidence and levels of fumonisin contamination in Colombian corn and corn products

A survey was conducted to determine the levels of fumonisins B1 and B2 in corn and corn-based products available in Colombia for human and animal consumption. A total of 120 samples were analyzed by acetonitrile-water extraction, cleanup with a strong-anion-exchange column, and liquid chromatography with o-phthaldialdehyde-2-mercaptoethanol derivatization and fluorescence detection. The samples of corn and corn-based products for animal intake were taken at different feed manufacturing plants, whereas the samples used for human foods where purchased from local retail stores. The number of positive samples for fumonisin B1 was 20.0% higher in corn and corn-based products for animal intake (75.0%) than in corn and corn-based products for human consumption (55.0%). The levels of fumonisin B1 were also higher in corn and corn-based products for animal intake (mean = 694 μg/kg; range = 32–2964 μg/kg), than in corn and corn-based products for human intake (mean = 218 μg/kg; range = 24–2170 μg/ kg). The incidence and levels of fumonisin B2 were lower than those for fumonisin B1. Corn and corn-based products for animal consumption had an incidence of fumonisin B2 of 58.3%, with a mean value of 283 μg/kg, and a range of 44–987 μg/kg. The incidence of fumonisin B2 in corn-based products for human intake was 35.0%, with a mean value of 118 μg/kg and a range of 21–833 μg/kg. The highest incidence and levels of fumonisins were found in samples of hominy feed, with concentrations ranging from 86 to 2964 μg/kg fumonisin B1 and 57 to 987 μg/kg fumonisin B2.     

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.     

Electrostatic calculations for an ion channel. II. Kinetic behavior of the gramicidin A channel.

A theoretical model of the gramicidin A channel is presented and the kinetic behavior of the model is derived and compared with previous experimental results. The major assumption of the model is that the only interaction between ions in a multiply-occupied channel is electrostatic. The electrostatic calculations indicate in a multiply-occupied channel is electrostatic. The electrostatic calculations indicate that there will be potential wells at each end of the channel and, at high concentrations, that both wells can be occupied. The kinetics are based on two reaction steps: movement of the ion from the bulk solution to the well and movement between the two wells. The kinetics for this reaction rate approach are identical to those based on the Nernst-Planck equation in the limit where the movement between the two wells is rate limiting. The experimental results for sodium and potassium are consistent with a maximum of two ions per channel. To explain the thallium results it is necessary to allow three ions per channel. It is shown that this case is compatible with the electrostatic calculations if the presence of an anion is included. The theoretical kinetics are in reasonable quantitative agreement with the following experimental measurements: single channel conductance of sodium, potassium, and thallium; bi-ionic potential and permeability ratio between sodium-potassium and potassium-thallium; the limiting conductance of potassium and thallium at high applied voltages; current-voltage curves for sodium and potassium at low (but not high) concentrations; and the inhibition of sodium conductance by thallium. The results suggest that the potential well is located close to the channel mouth and that the conductance is partially limited by the rate going from the bulk solution to the well. For thallium, this entrance rate is probably diffusion limited.     

Strong electrolyte continuum theory solution for equilibrium profiles, diffusion limitation, and conductance in charged ion channels.

The solution for the ion flux through a membrane channel that incorporates the electrolyte nature of the aqueous solution is a difficult theoretical problem that, until now, has not been properly formulated. The difficulty arises from the complicated electrostatic problem presented by a high dielectric aqueous channel piercing a low dielectric lipid membrane. The problem is greatly simplified by assuming that the ratio of the dielectric constant of the water to that of the lipid is infinite. It is shown that this is a good approximation for most channels of biological interest. This assumption allows one to derive simple analytical expressions for the Born image potential and the potential from a fixed charge in the channel, and it leads to a differential equation for the potential from the background electrolyte. This leads to a rigorous solution for the ion flux or the equilibrium potential based on a combination of the Nernst-Planck equation and strong electrolyte theory (i.e., Gouy-Chapman or Debye-Huckel). This approach is illustrated by solving the system of equations for the specific case of a large channel containing fixed negative charges. The following characteristics of this channels are discussed: anion and mono- and divalent cation conductance, saturation of current with increasing concentration, current-voltage relationship, influence of location and valence of fixed charge, and interaction between ions. The qualitative behavior of this channel is similar to that of the acetylcholine receptor channel.