Aryl urea analogs with broad-spectrum antibacterial activity

The preparation and evaluation of novel aryl urea analogs as broad-spectrum antibacterial agents is described. Numerous compounds showed low micromolar minimum inhibitory concentrations (MIC) against both Gram-positive and Gram-negative bacteria. Selected analogs also exhibited in vivo efficacy in a lethal murine model of bacterial septicemia.     

Sphingolipid storage induces accumulation of intracellular cholesterol by stimulating SREBP-1 cleavage

We showed previously that the intracellular transport of sphingolipids (SLs) is altered in SL storage disease fibroblasts, due in part to the secondary accumulation of free cholesterol. In the present study we examined the mechanism of cholesterol elevation in normal human skin fibroblasts induced by treatment with SLs. When cells were incubated with various natural SLs for 44 h, cholesterol levels increased 25-35%, and cholesterol esterification was reduced. Catabolism of the exogenous SLs was not required for elevation of cholesterol because (i) a non-hydrolyzable and a degradable SL analog elevated cellular cholesterol to similar extents, and (ii) incubation of cells with various SL catabolites, including ceramide, had no effect on cholesterol levels. Elevated cholesterol was derived primarily from low density lipoproteins (LDL) and resulted from up-regulation of LDL receptors induced by cleavage of the sterol regulatory element-binding protein-1. Upon SL treatment, cholesterol accumulated with exogenous SLs in late endosomes and lysosomes. These results suggest a model in which excess SLs present in endocytic compartments serve as a "molecular trap" for cholesterol, leading to a reduction in cholesterol at the endoplasmic reticulum, induction of sterol regulatory element-binding protein-1 cleavage, and up-regulation of LDL receptors.     

Integration of hypoxic dilation signaling pathways for skeletal muscle resistance arteries

Mediator contributions to hypoxic dilation of rat gracilis muscle resistance arteries were determined by measuring dilation, vascular smooth muscle hyperpolarization, and metabolite production after incremental hypoxia. Nitric oxide (NO) synthase inhibition abolished responses to mild hypoxia, whereas COX inhibition impaired responses to more severe hypoxia by 77%. Blocking 20-hydroxyeicosatetraenoic acid (20-HETE) impaired responses to moderate hypoxia. With only NO systems intact, responses were maintained with mild hypoxia (88% normal) mediated via K(Ca) channels. When only COX pathways were intact, responses to moderate-severe hypoxia were largely retained (79% of normal) mediated via K(ATP) channels. Vessel responses to moderate hypoxia were retained with only 20-HETE systems intact mediated via K(Ca) channels. NO production increased 5.6-fold with mild hypoxia; greater hypoxia was without further effect. With increased hypoxia, 20-HETE levels fell to 40% of control values. 6-keto-PGF(1alpha) levels were not altered with mild hypoxia, but increased 4.6-fold with severe hypoxia. These results suggest vascular reactivity to progressive hypoxia represents an integration of NO production (mild hypoxia), PGI(2) production (severe hypoxia), and reduced 20-HETE levels (moderate hypoxia).     

Formation of a copper specific binding site in non-native states of beta-2-microglobulin

A debilitating complication of long-term hemodialysis is the deposition of beta-2-microglobulin (beta2m) as amyloid plaques in the joint space. We have recently shown that Cu(2+) can be a contributing, if not causal, factor at concentrations encountered during dialysis therapy. The basis for this effect is destabilization and incorporation of beta2m into amyloid fibers upon binding of Cu(2+). In this work, we demonstrate that while beta2m binds Cu(2+) specifically in the native state, it is binding of Cu(2+) by non-native states of beta2m which is responsible for destabilization. Mutagenesis of potential coordinating groups for Cu(2+) shows that native state binding of Cu(2+) is mediated by residues and structures that are different than those which bind in non-native states. An increased affinity for copper by non-native states compared to that of the native state gives rise to overall destabilization. Using mass spectrometry, NMR, and fluorescence techniques, we show that native state binding is localized to H31 and W60 and is highly specific for Cu(2+) over Zn(2+) and Ni(2+). Binding of Cu(2+) in non-native states of beta2m is mediated by residues H13, H51, and H84, but not H31. Although denatured beta2m has characteristics of a globally unfolded state, it nevertheless demonstrates the following strong specificity of binding: Cu(2+) > Zn(2+) > Ni(2+). This requires the existence of a well-defined structure in the unfolded state of this protein. As Cu(2+) effects are reported in many other amyloidoses, e.g., PrP, alpha-synuclein, and Abeta, our results may be extended to the emerging field of divalent ion-associated amyloidosis.     

"Superhumanized" antibodies: reduction of immunogenic potential by complementarity-determining region grafting with human germline sequences: application to an anti-CD28

Humanized Abs are created by combining, at the genetic level, the complementarity-determining regions of a murine mAb with the framework sequences of a human Ab variable domain. This leads to a functional Ab with reduced immunogenic side effects in human therapy. In this study, we report a new approach to humanizing murine mAbs that may reduce immunogenicity even further. This method is applied to humanize the murine anti-human CD28 Ab, 9.3. The canonical structures of the hypervariable loops of murine 9.3 were matched to human genomic V gene sequences whose hypervariable loops had identical or similar canonical structures. Framework sequences for those human V genes were then used, unmodified, with the 9.3 complementarity-determining regions to construct a humanized version of 9.3. The humanized 9.3 and a chimeric 9.3 control were expressed in Escherichia coli as Fab. The humanized Fab showed a moderate loss in avidity in a direct binding ELISA with immobilized CD28-Ig fusion protein (CD28-Ig). Humanized 9.3 blocked ligation of CD28-Ig to cells expressing the CD28 receptor CD80. Lastly, the humanized 9.3 showed biological activity as an immunosuppressant by inhibiting a MLR.     

Developmental decline in components of signal transduction pathways regulating protein synthesis in pig muscle

Our previous studies showed that the feeding-induced stimulation of protein synthesis in skeletal muscle of neonatal pigs is accompanied by enhanced phosphorylation of the eukaryotic initiation factor (eIF)4E-binding protein (4E-BP1) and the ribosomal protein S6 kinase (S6K1). These effects of feeding are substantially reduced with development. The goal of the present investigation was to delineate the basis for the reduced responsiveness to feeding observed in the older animals. In these studies, the content and activity of protein kinases located upstream of S6K1 and 4E-BP1 in signal transduction pathways activated by amino acids, insulin, and insulin-like growth factor I were examined in 7- and 26-day-old pigs that were either fasted overnight or fed porcine milk after an overnight fast. Feeding stimulated phosphatidylinositol (PI) 3-kinase activity to the same extent in muscle of 7- and 26-day-old pigs, suggesting that PI 3-kinase is not limiting in muscle of older animals. In contrast, protein kinase B (PKB) activity was significantly less in muscle from 26- vs. 7-day-old pigs, regardless of nutritional status, suggesting that its activity is regulated by mechanisms distinct from PI 3-kinase. In part, the reduced PKB responsiveness can be attributed to a developmental decline in PKB content. Likewise, muscle content of the protein kinase termed mammalian target of rapamycin (mTOR) in 26-day-old pigs was <25% of that in 7-day-old animals. Finally, in agreement with our earlier work showing that S6K1 phosphorylation is reduced in older animals, S6K1 activity was stimulated to a lesser extent in 26- compared with 7-day-old pigs. Overall, the results suggest that the blunted protein synthetic response observed in 26- vs. 7-day-old neonatal pigs is due in part to decreased content and/or activity of signaling components downstream of PI 3-kinase, e.g., PKB, mTOR, and S6K1.     

Contribution of insulin to the translational control of protein synthesis in skeletal muscle by leucine

Enhanced protein synthesis in skeletal muscle after ingestion of a balanced meal in postabsorptive rats is mimicked by oral leucine administration. To assess the contribution of insulin to the protein synthetic response to leucine, food-deprived (18 h) male rats (approximately 200 g) were intravenously administered a primed-constant infusion of somatostatin (60 microg + 3 microg.kg(-1).h(-1)) or vehicle beginning 1 h before administration of leucine (1.35 g L-leucine/kg) or saline (control). Rats were killed 15, 30, 45, 60, or 120 min after leucine administration. Compared with controls, serum insulin concentrations were elevated between 15 and 45 min after leucine administration but returned to basal values by 60 min. Somatostatin maintained insulin concentrations at basal levels throughout the time course. Protein synthesis was increased between 30 and 60 min, and this effect was blocked by somatostatin. Enhanced assembly of the mRNA cap-binding complex (composed of eukaryotic initiation factors eIF4E and eIF4G) and hyperphosphorylation of the eIF4E-binding protein 1 (4E-BP1), the 70-kDa ribosomal protein S6 kinase (S6K1), and the ribosomal protein S6 (rp S6) were observed as early as 15 min and persisted for at least 60 min. Somatostatin attenuated the leucine-induced changes in 4E-BP1 and S6K1 phosphorylation and completely blocked the change in rp S6 phosphorylation but had no effect on eIF4G small middle dot eIF4E assembly. Overall, the results suggest that the leucine-induced enhancement of protein synthesis and the phosphorylation states of 4E-BP1 and S6K1 are facilitated by the transient increase in serum insulin. In contrast, assembly of the mRNA cap-binding complex occurs independently of increases in insulin and, by itself, is insufficient to stimulate rates of protein synthesis in skeletal muscle after leucine administration.     

Measuring kinesin's first step

A variety of models have recently emerged to explain how the molecular motor kinesin is able to maintain processive movement for over 100 steps. Although these models differ in significant features, they all predict that kinesin's catalytic domains intermittently separate from each other as the motor takes 8-nm steps along the microtubule. Furthermore, at some point in this process, one molecule of ATP is hydrolyzed per step. However, exactly when hydrolysis and product release occur in relation to this forward step have not been established. Furthermore, the rate at which this separation occurs as well as the speed of motor stepping onto and release from the microtubule have not been measured. In the absence of this information, it is difficult to critically evaluate competing models of kinesin function. We have addressed this issue by developing spectroscopic probes whose fluorescence is sensitive to motor-motor separation or microtubule binding. The kinetics of these fluorescence changes allow us to directly measure how fast kinesin steps onto and releases from the microtubule and provide insight into how processive movement is maintained by this motor.     

Genomic studies of gene expression: regulation of the Wilson disease gene

Bacterial artificial chromosomes (BACs) have many advantages over other large-insert cloning vectors and have been used for a variety of genetic applications, including the final contigs of the human genome. We describe the utilization of a BAC construct to study gene regulation in a tissue culture-based system, using a 170-kb clone containing the entire Wilson disease (WND) locus as a model. A second BAC construct that lacked a putative negatively regulating promoter sequence was created. A nonviral method of gene delivery was applied to transfect three human cell lines stably with each construct. Our results show correct WND gene expression from the recombinant locus and quantification revealed significantly increased expression from the clone lacking the negative regulator. Comparison with conventional methods confirms the reliability of the genomic approach for thorough examination of gene expression. This experimental system illustrates the potential of BAC clones in genomic gene expression studies, new gene therapy strategies, and validation of potential molecular targets for drug discovery.     

4E-BP1 and S6K1: translational integration sites for nutritional and hormonal information in muscle

Maintenance of cellular protein stores in skeletal muscle depends on a tightly regulated synthesis-degradation equilibrium that is conditionally modulated under an extensive range of physiological and pathophysiological circumstances. Recent studies have established the initiation phase of mRNA translation as a pivotal site of regulation for global rates of protein synthesis, as well as a site through which the synthesis of specific proteins is controlled. The protein synthetic pathway is exquisitely sensitive to the availability of hormones and nutrients and employs a comprehensive integrative strategy to interpret the information provided by hormonal and nutritional cues. The translational repressor, eukaryotic initiation factor 4E binding protein 1 (4E-BP1), and the 70-kDa ribosomal protein S6 kinase (S6K1) have emerged as important components of this strategy, and together they coordinate the behavior of both eukaryotic initiation factors and the ribosome. This review discusses the role of 4E-BP1 and S6K1 in translational control and outlines the mechanisms through which hormones and nutrients effect changes in mRNA translation through the influence of these translational effectors.