Some Physicochemical Properties of Invertase of Candida utilis

This study examined the microbial transformation of carbazole (CZ) by an isolated bacterium that can use CZ as a sole carbon and nitrogen source. The strain identified as Pseudomonas stutzeri produced a large amount of anthranilic acid (AA) from CZ in the medium containing a nonionic surfactant. In dialysis culture using ion-exchange resin, 7.9 g/liter (58mm) of AA was accumulated from 15g/liter (90mm) of CZ and the molar yield of AA reached about 64%.     

HIV gp120- and methamphetamine-mediated oxidative stress induces astrocyte apoptosis via cytochrome P450 2E1

HIV-1 glycoprotein 120 (gp120) is known to cause neurotoxicity via several mechanisms including production of proinflammatory cytokines/chemokines and oxidative stress. Likewise, drug abuse is thought to have a direct impact on the pathology of HIV-associated neuroinflammation through the induction of proinflammatory cytokines/chemokines and oxidative stress. In the present study, we demonstrate that gp120 and methamphetamine (MA) causes apoptotic cell death by inducing oxidative stress through the cytochrome P450 (CYP) and NADPH oxidase (NOX) pathways. The results showed that both MA and gp120 induced reactive oxygen species (ROS) production in concentration- and time-dependent manners. The combination of gp120 and MA also induced CYP2E1 expression at both mRNA (1.7±0.2- and 2.8±0.3-fold in SVGA and primary astrocytes, respectively) and protein (1.3±0.1-fold in SVGA and 1.4±0.03-fold in primary astrocytes) levels, suggesting the involvement of CYP2E1 in ROS production. This was further confirmed by using a selective inhibitor of CYP2E1, diallylsulfide (DAS), and CYP2E1 knockdown using siRNA, which significantly reduced ROS production (30–60%). As the CYP pathway is known to be coupled with the NOX pathway, including Fenton–Weiss–Haber (FWH) reaction, we examined whether the NOX pathway is also involved in ROS production induced by either gp120 or MA. Our results showed that selective inhibitors of NOX, diphenyleneiodonium (DPI), and FWH reaction, deferoxamine (DFO), also significantly reduced ROS production. These findings were further confirmed using specific siRNAs against NOX2 and NOX4 (NADPH oxidase family). We then showed that gp120 and MA both induced apoptosis (caspase-3 activity and DNA lesion using TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling) assay) and cell death. Furthermore, we showed that DAS, DPI, and DFO completely abolished apoptosis and cell death, suggesting the involvement of CYP and NOX pathways in ROS-mediated apoptotic cell death. In conclusion, this is the first report on the involvement of CYP and NOX pathways in gp120/MA-induced oxidative stress and apoptotic cell death in astrocytes, which has clinical implications in neurodegenerative diseases, including neuroAIDS.     

Application of a new human cell line,F2N78, in the transient and stable production of recombinant therapeutics

Host cell lines developed by genetic engineering sometimes show instabilities in maintaining their genetically acquired phenotypes. Previously, a hybrid host cell line, designated as hybrid of kidney and B cells (HKB), capable of retaining selected phenotypes originally existing in the parental cells was developed via fusion of 293 cells and HH514‐16 cells. Although HKB did indeed successfully preserve several favorable phenotypes, the expression of Epstein‐Barr virus (EBV) specific nuclear antigen 1 (EBNA1), which should be constitutively expressed for host cells to utilize oriP expression vector in transient production of therapeutic proteins, was observed to be unstable. Here, in an attempt to obtain stable expression of EBNA1, a cell type that contains an integrated EBV genome, rather than HH514‐16 cells, which harbor an episomal EBV genome, was applied for fusion with 293 cells. Fusion of 293 cells with Namalwa cells led to the creation of a new type of hybrid, F2N, which was able to stably express EBNA1 while not producing EBV particles. One of the F2N clones, F2N78, was observed to maintain EBNA1 expression for more than 1 year under serum‐free suspension culture conditions along with human specific glycosyl phenotypes observed previously in HKB. In addition, F2N78 was demonstrated to be an appropriate host cell line for both the transient and stable production of recombinant therapeutics with the features of safety expected of production cell lines for human use. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29: 432–440, 2013     

A high throughput substrate binding assay reveals hexachlorophene as an inhibitor of the ER-resident HSP70 chaperone GRP78

Glucose-regulated protein 78 (GRP78) is the ER resident 70 kDa heat shock protein 70 (HSP70) and has been hypothesized to be a therapeutic target for various forms of cancer due to its role in mitigating proteotoxic stress in the ER, its elevated expression in some cancers, and the correlation between high levels for GRP78 and a poor prognosis. Herein we report the development and use of a high throughput fluorescence polarization-based peptide binding assay as an initial step toward the discovery and development of GRP78 inhibitors. This assay was used in a pilot screen to discover the anti-infective agent, hexachlorophene, as an inhibitor of GRP78. Through biochemical characterization we show that hexachlorophene is a competitive inhibitor of the GRP78-peptide interaction. Biological investigations showed that this molecule induces the unfolded protein response, induces autophagy, and leads to apoptosis in a colon carcinoma cell model, which is known to be sensitive to GRP78 inhibition.     

Modeling interaction between gp120 HIV protein and CCR5 receptor

The study of the process of HIV entry into the host cell and the creation of biomimetic nanosystems that are able to selectively bind viral particles and proteins is a high priority research area for the development of novel diagnostic tools and treatment of HIV infection. Recently, we described multilayer nanoparticles (nanotraps) with heparin surface and cationic peptides comprising the N‐terminal tail (Nt) and the second extracellular loop (ECL2) of CCR5 receptor, which could bind with high affinity some inflammatory chemokines, in particular, Rantes. Because of the similarity of the binding determinants in CCR5 structure, both for chemokines and gp120 HIV protein, here we expand this approach to the study of the interactions of these biomimetic nanosystems and their components with the peptide representing the V3 loop of the activated form of gp120. According to surface plasmon resonance results, a conformational rearrangement is involved in the process of V3 and CCR5 fragments binding. As in the case of Rantes, ECL2 peptide showed much higher affinity to V3 peptide than Nt (K_D = 3.72 × 10^?8 and 1.10 × 10^?6 M, respectively). Heparin‐covered nanoparticles bearing CCR5 peptides effectively bound V3 as well. The presence of both heparin and the peptides in the structure of the nanotraps was shown to be crucial for the interaction with the V3 loop. Thus, short cationic peptides ECL2 and Nt proved to be excellent candidates for the design of CCR5 receptor mimetics.     

Functional mimetic of the G-protein coupled receptor CXCR4 on a soluble antibody scaffold

G-protein coupled receptors (GPCRs) constitute major drug targets due to their involvement in critical biological functions and pathophysiological disorders. The leading challenge in their structural and functional characterization has been the need for a lipid environment to accommodate their hydrophobic cores. Here, we report an antibody scaffold mimetic (ASM) platform where we have recapitulated the extracellular functional domains of the GPCR, C-X-C chemokine receptor 4 (CXCR4) on a soluble antibody framework. The engineered ASM molecule can accommodate the N-terminal loop and all three extracellular loops of CXCR4. These extracellular features are important players in ligand recruitment and interaction for allostery and signal transduction. Our study shows that ASM^CXCR4 can be recognized by the anti-CXCR4 antibodies, MEDI3185, 2B11, and 12G5, and that ASM^CXCR4 can bind the HIV-1 glycoprotein ligand gp120, and the natural chemokine ligand SDF-1α. Further, we show that ASM^CXCR4 can competitively inhibit the SDF-1α signaling pathway, and be used as an immunogen to generate CXCR4-specific antibodies. This platform will be useful in the study of GPCR biology in a soluble receptor context for evaluating its extracellular ligand interactions.     

Rapid, transient induction of ER stress in the liver and kidney after acute exposure to heavy metal: evidence from transgenic sensor mice

Endoplasmic reticulum (ER) stress-responsive alkaline phosphatase (ES-TRAP) serves as a sensitive indicator for ER stress. In response to heavy metals including cadmium, nickel and cobalt, hepatocytes and renal tubular cells expressing ES-TRAP exhibited ER stress and decreased ES-TRAP activity. In ES-TRAP transgenic mice, acute exposure to cadmium showed rapid, transient decreases in the activity of serum ES-TRAP. It was inversely correlated with the induction of endogenous ER stress markers in the liver and kidney. Our result provides first evidence for the acute, reversible induction of ER stress in vivo after exposure to heavy metal.     

Dynamic domains and geometrical properties of HIV-1 gp120 during conformational changes induced by CD4 binding

The HIV-1 gp120 exterior envelope glycoprotein undergoes a series of conformational rearrangements while sequentially interacting with the receptor CD4 and coreceptor CCR5 or CXCR4 on the surface of host cells to initiate virus entry. Both the crystal structures of the HIV-1 gp120 core bound by the CD4 and antigen 17b, and the SIV gp120 core pre-bound by the CD4 are known. We have performed dynamic domain studies on the homology models of the CD4-bound and unliganded HIV-1 gp120 with modeled V3 and V4 loops to explore details of conformational changes, hinge axes, and hinge bending regions in the gp120 structures upon CD4 binding. Four dynamic domains were clustered and intricately motional modes for domain pairs were discovered. Together with the detailed comparative analyses of geometrical properties between the unliganded and liganded gp120 models, an induced fit model was proposed to explain events accompanying the CD4 engagement to the gp120, which provided new insight into the dynamics of the molecular induced binding mechanism that complements the molecular dynamics and crystallographic studies.     

CCR5 interactions with the variable 3 loop of gp120

The G-protein coupled receptor CCR5 functions pathologically as the primary co-receptor for macrophage tropic (R5) strains of HIV-1. The interactions responsible for co-receptor activity are unknown. Molecular-dynamics simulations of the extracellular and adjacent transmembrane domains of CCR5 were performed with explicit solvation utilizing a rhodopsin-based homology model. The functional unit of co-receptor binding was constructed via docking and molecular-dynamics simulation of CCR5 and the variable 3 loop of gp120, which is a dominant determinant of co-receptor utilization. The variable 3 loop was demonstrated to interact primarily with the amino terminus and the second extracellular loop of CCR5, providing novel structural information regarding the co-receptor-binding site. Alanine mutants that alter chemokine binding and co-receptor activity were examined. Molecular-dynamics simulations with and without the variable 3 loop of gp120 were able to rationalize the activities of these mutants successfully, providing support for the proposed model. Based on these results, the global complex of CCR5, gp120 including the V3 loop and CD4, was investigated. The utilization of computational analysis, in combination with molecular biological data, provides a powerful approach for understanding the use of CCR5 as a co-receptor by HIV-1.