Search

Aristolochic acids (AAs) are naturally occurring nitro phenanthrene carboxylic acids primarily found in plants of the Aristolochiaceae family. Aristolochic acid D (AAD) is a major constituent in the roots and rhizomes of the Chinese herb Xixin (the roots and rhizomes of Asarum heterotropoides F. Schmidt), which is a key material for preparing a suite of marketed Chinese medicines. Structurally, AAD is nearly identical to the nephrotoxic aristolochic acid I (AAI), with an additional phenolic group at the C-6 site. Although the nephrotoxicity and metabolic pathways of AAI have been well-investigated, the metabolic pathway(s) of AAD in humans and the influence of AAD metabolism on its nephrotoxicity has not been investigated yet. To identify the major metabolites of AAD in human tissues and to characterize AAD O -glucuronidation kinetics in different enzyme sources, as well as to explore the influence of AAD O -glucuronidation on its nephrotoxicity. The O -glucuronide of AAD was biosynthesized and its chemical structure was fully characterized by both 1H-NMR and 13C-NMR. Reaction phenotyping assays, chemical inhibition assays, and enzyme kinetics analyses were conducted to assess the crucial enzymes involved in AAD O -glucuronidation in humans. Docking simulations were performed to mimic the catalytic conformations of AAD in human UDP-glucuronosyltransferases (UGTs), while the predicted binding energies and distances between the deprotonated C-6 phenolic group of AAD and the glucuronyl moiety of UDPGA in each tested human UGT isoenzyme were measured. The mitochondrial membrane potentials (MMP) and reactive oxygen species (ROS) levels in HK-2 cells treated with either AAI, or AAD, or AAD O -glucuronide were tested, to elucidate the impact of O -glucuronidation on the nephrotoxicity of AAD. AAD could be rapidly metabolized in human liver and intestinal microsomes (HLM and HIM, respectively) to form a mono-glucuronide, which was purified and fully characterized as AAD-6- O -β-D-glucuronide (AADG) by NMR. UGT1A1 was the predominant enzyme responsible for AAD-6- O -glucuronidation, while UGT1A9 contributed to a lesser extent. AAD-6- O -glucuronidation in HLM, HIM, UGT1A1 and UGT1A9 followed Michaelis-Menten kinetics, with the K m values of 4.27 μM, 9.05 μM, 3.87 μM, and 7.00 μM, respectively. Docking simulations suggested that AAD was accessible to the catalytic cavity of UGT1A1 or UGT1A9 and formed catalytic conformations. Further investigations showed that both AAI and AAD could trigger the elevated intracellular ROS levels and induce mitochondrial dysfunction and in HK-2 cells, but AADG was hardly to trigger ROS accumulation and mitochondrial dysfunction. Collectively, UGT1A-catalyzed AAD 6- O -glucuronidation represents a crucial detoxification pathway of this naturally occurring AAI analogs in humans, which is very different from that of AAI. Aristolochic acid D (AAD), an abundant naturally occurring aristolochic acid derivative isolated from the Chinese herb Xixin (the roots and rhizomes of Asarum heterotropoides F. Schmidt), can be rapidly converted into AAD-6- O -β-D-glucuronide (AADG) in human tissue preparations. UGT1A-catalyzed AAD 6- O -glucuronidation offers a crucial detoxification pathway of this aristolochic acid derivative in humans, which is very different from that of the nephrotoxin aristolochic acid I (AAI). [Display omitted] • AAD- O -glucuronidation was a major metabolic pathway of AAD in humans. • The glucuronide was biosynthesized and characterized as AAD-6- O -β-D-glucuronide. • UGT1A1 was the predominant enzyme responsible for AAD-6- O -glucuronidation. • AAD-6- O -glucuronidation offers a crucial detoxification pathway of AAD in humans. [ABSTRACT FROM AUTHOR]

Abstract: Designed zinc finger proteins (ZFPs) regulate expression of target genes when coupled to activator or repressor domains. Transfection of ZFPs into cell lines can create expression systems where the targeted endogenous gene is transcribed and the protein of interest can be investigated in its own cellular context. Here we describe the pharmacological investigation of an expression system generated using CCK2 receptor-selective ZFPs transfected into human embryonic kidney cells (HEKZFP system). The receptors expressed in this system, in response to ZFP expression, were functional in calcium mobilization studies and the potency of the agonists investigated was consistent with their action at CCK2 receptors (CCK-8S pA50 =9.05±0.11, pentagastrin pA50 =9.11±0.13). In addition, binding studies were conducted using [125I]-BH-CCK-8S as radioligand. The saturation binding analysis of this radioligand was consistent with a single population of high affinity CCK receptors (pKD =10.24). Competition studies were also conducted using a number of previously well-characterized CCK-receptor selective ligands; JB93182, YF476, PD-134,308, SR27897, dexloxiglumide, L-365,260 and L-364,718. Overall, the estimated affinity values for these ligands were consistent with their interaction at CCK2 receptors. Therefore, CCK2 receptors up-regulated using zinc finger protein technology can provide an alternative to standard transfection techniques for the pharmacological analysis of compounds. [Copyright &y& Elsevier]

Focuses on a study which shows that integral membrane protein HisP can be separated from HisQ and HisM and reassembled back into an active HisQMP2 complex. Materials and methods used; Indepth look at the superfamily of ABC transporters; Results and discussion.

Hydrogen storage steel cylinders are the earliest and widely used hydrogen storage vessels. Fatigue cracks are easy to initiate and grow under hydrogen pressure, which threatens the safety of users. Although hydrogen has an effect on the initiation and growth of fatigue cracks, a reasonable structure of cylinder will make the stress distribution more reasonable and reduce the probability of crack initiation from the source. This paper researched the multi-center concave bottom for hydrogen storage cylinder. The geometric design parameters of the new base end are determined by the orthogonal design method. We analyze the effects of various parameters on the multi-center concave base end by using the finite element method. Based on the finite element analysis (FEA) of different structure, the results show that the stress concentration of new base end can be greatly reduced, and the minimum stress concentration factor is close to 1.1. The results provide valuable insights for designing and manufacturing the new type of seamless gas cylinder. The corresponding gas cylinder that we processed according to the simulation had successfully passed a series of tests. • Using orthogonal design method to determine hydrogen cylinders' parameters. • Analysis of stress of hydrogen storage cylinders by finite element method. • Newly designed hydrogen storage cylinders have excellent performance. • The manufactured hydrogen storage cylinders have passed the test verification. [ABSTRACT FROM AUTHOR]

In order to solve the inconsistency problem of the theoretical solutions of critical buckling temperature rise for thin cylindrical shells reported in the existing literatures, a first attempt was made in the present study to perform a derivation process on the critical internal force, thermal stress and critical buckling temperature rise for the rectangular thin plate and thin cylindrical shell based on small deformation theory and the Donnell form of the nonlinear equilibrium equations, respectively. Thereafter, the theoretical solutions of the thermal stresses under different boundary conditions and temperature rise variations were determined. The results show that the theoretical solutions of the internal force, thermal stress and critical buckling temperature rise are in good agreement with the numerical results. Finally, the reason leading to the inconsistency of the theoretical solution of the critical buckling temperature rise was elucidated in detail. [ABSTRACT FROM AUTHOR]

Regulatory regions harbor multiple transcription factor (TF) recognition sites; however, the contribution of individual sites to regulatory function remains challenging to define. We describe an approach that exploits the error-prone nature of genome editing-induced double-strand break repair to map functional elements within regulatory DNA at nucleotide resolution. We demonstrate the approach on a human erythroid enhancer, revealing single TF recognition sites that gate the majority of downstream regulatory function. [ABSTRACT FROM AUTHOR]

The HIV-1 coreceptor CCR5 is a validated target for HIV/AIDS therapy. The apparent elimination of HIV-1 in a patient treated with an allogeneic stem cell transplant homozygous for a naturally occurring CCR5 deletion mutation (CCR5Δ32/Δ32) supports the concept that a single dose of HIV-resistant hematopoietic stem cells can provide disease protection. Given the low frequency of naturally occurring CCR5Δ32/Δ32 donors, we reasoned that engineered autologous CD34+ hematopoietic stem/progenitor cells (HSPCs) could be used for AIDS therapy. We evaluated disruption of CCR5 gene expression in HSPCs isolated from granulocyte colony-stimulating factor (CSF)-mobilized adult blood using a recombinant adenoviral vector encoding a CCR5-specific pair of zinc finger nucleases (CCR5-ZFN). Our results demonstrate that CCR5-ZFN RNA and protein expression from the adenoviral vector is enhanced by pretreatment of HSPC with protein kinase C (PKC) activators resulting in >25% CCR5 gene disruption and that activation of the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathway is responsible for this activity. Importantly, using an optimized dose of PKC activator and adenoviral vector we could generate CCR5-modified HSPCs which engraft in a humanized mouse model (albeit at a reduced level) and support multilineage differentiation in vitro and in vivo. Together, these data establish the basis for improved approaches exploiting adenoviral vector delivery in the modification of HSPCs. [ABSTRACT FROM AUTHOR]

The transfer of high-avidity T cell receptor (TCR) genes isolated from rare tumor-specific lymphocytes into polyclonal T cells is an attractive cancer immunotherapy strategy. However, TCR gene transfer results in competition for surface expression and inappropriate pairing between the exogenous and endogenous TCR chains, resulting in suboptimal activity and potentially harmful unpredicted antigen specificities of the resultant TCRs. We designed zinc-finger nucleases (ZFNs) that promoted the disruption of endogenous TCR ?- and ?-chain genes. Lymphocytes treated with ZFNs lacked surface expression of CD3-TCR and expanded with the addition of interleukin-7 (IL-7) and IL-15. After lentiviral transfer of a TCR specific for the Wilms tumor 1 (WT1) antigen, these TCR-edited cells expressed the new TCR at high levels, were easily expanded to near purity and were superior at specific antigen recognition compared to donor-matched, unedited TCR-transferred cells. In contrast to unedited TCR-transferred cells, the TCR-edited lymphocytes did not mediate off-target reactivity while maintaining their anti-tumor activity in vivo, thus showing that complete editing of T cell specificity generates tumor-specific lymphocytes with improved biosafety profiles. [ABSTRACT FROM AUTHOR]

Human induced pluripotent stem cells (iPSCs) represent a unique opportunity for regenerative medicine because they offer the prospect of generating unlimited quantities of cells for autologous transplantation, with potential application in treatments for a broad range of disorders. However, the use of human iPSCs in the context of genetically inherited human disease will require the correction of disease-causing mutations in a manner that is fully compatible with clinical applications. The methods currently available, such as homologous recombination, lack the necessary efficiency and also leave residual sequences in the targeted genome. Therefore, the development of new approaches to edit the mammalian genome is a prerequisite to delivering the clinical promise of human iPSCs. Here we show that a combination of zinc finger nucleases (ZFNs) and piggyBac technology in human iPSCs can achieve biallelic correction of a point mutation (Glu342Lys) in the ?1-antitrypsin (A1AT, also known as SERPINA1) gene that is responsible for ?1-antitrypsin deficiency. Genetic correction of human iPSCs restored the structure and function of A1AT in subsequently derived liver cells in vitro and in vivo. This approach is significantly more efficient than any other gene-targeting technology that is currently available and crucially prevents contamination of the host genome with residual non-human sequences. Our results provide the first proof of principle, to our knowledge, for the potential of combining human iPSCs with genetic correction to generate clinically relevant cells for autologous cell-based therapies. [ABSTRACT FROM AUTHOR]