Human Embryo Kidney Research Articles

Influence of chitosan nanofiber scaffold on porcine endogenous retroviral expression and infectivity in pig hepatocytes

To investigate the influence of chitosan nanofiber scaffold on the production and infectivity of porcine endogenous retrovirus (PERV) expressed by porcine hepatocytes. Freshly isolated porcine hepatocytes were cultured with or without chitosan nanofiber scaffold (defined as Nano group and Hep group) for 7 d. The daily collection of culture medium was used to detect reverse transcriptase (RT) activity with RT activity assay kits and PERV RNA by reverse transcription-polymerase chain reaction (PCR) and real time PCR with the PERV specific primers. And Western blotting was performed with the lysates of daily retrieved cells to determine the PERV protein gag p30. Besides, the in-vitro infectivity of the supernatant was tested by incubating the human embryo kidney 293 (HEK293) cells. The similar changing trends between two groups were observed in real time PCR, RT activity assay and Western blotting. Two peaks of PERV expression at 10H and Day 2 were found and followed by a regular decline. No significant difference was found between two groups except the significantly high level of PERV RNA at Day 6 and PERV protein at Day 5 in Nano group than that in Hep group. And in the in-vitro infection experiment, no HEK293 cell was infected by the supernatant. Chitosan nanofiber scaffold might prolong the PERV secreting time in pig hepatocytes but would not obviously influence its productive amount and infectivity, so it could be applied in the bioartificial liver without the increased risk of the virus transmission.

Estrogen regulation of human with-no-lysine (K) kinase-4 gene expression involves AP-1 transcription factor

With-no-lysine (K) kinase-4 (WNK4) is a serine/threonine protein kinase and plays a crucial role in the regulation of blood pressure and electrolyte homeostasis. Whether or not estrogen, an important regulator of physiologic functions, modulates human WNK4 (hWNK4) gene expression is unknown. In the current study, real-time PCR assay showed that 17β-estradiol (E2) with estrogen receptor alpha (ERα) suppressed the level of hWNK4 mRNA in cultured human embryo kidney 293 cells (HEK293). Luciferase activity analysis of the truncated hWNK4 promoters indicated that a regulatory region from −216 to −202 is essential for the basal transcriptional activity and response to E2/ERα. Using an electrophoresis mobility shift assay and a chromatin immunoprecipitation assay, we identified an activator protein-1 (AP-1) element at position −215/−205, to which AP-1 binding was enhanced by E2/ERα. Accordingly, AP-1 protein was increased by E2/ERα using Western blot analysis. Moreover, re-chromatin-immunoprecipitation and co-immunoprecipitation assays showed a direct interaction between ERα and AP-1 in HEK293 cells. In summary, these data document that E2/ERα regulates hWNK4 expression partly through AP-1 binding to the hWNK4 promoter.

A 24-Residue Peptide (p5), Derived from p35, the Cdk5 Neuronal Activator, Specifically Inhibits Cdk5-p25 Hyperactivity and Tau Hyperphosphorylation

The activity of Cdk5-p35 is tightly regulated in the developing and mature nervous system. Stress-induced cleavage of the activator p35 to p25 and a p10 N-terminal domain induces deregulated Cdk5 hyperactivity and perikaryal aggregations of hyperphosphorylated Tau and neurofilaments, pathogenic hallmarks in neurodegenerative diseases, such as Alzheimer disease and amyotrophic lateral sclerosis, respectively. Previously, we identified a 125-residue truncated fragment of p35 called CIP that effectively and specifically inhibited Cdk5-p25 activity and Tau hyperphosphorylation induced by Aβ peptides in vitro, in HEK293 cells, and in neuronal cells. Although these results offer a possible therapeutic approach to those neurodegenerative diseases assumed to derive from Cdk5-p25 hyperactivity and/or Aβ induced pathology, CIP is too large for successful therapeutic regimens. To identify a smaller, more effective peptide, in this study we prepared a 24-residue peptide, p5, spanning CIP residues Lys(245)-Ala(277). p5 more effectively inhibited Cdk5-p25 activity than did CIP in vitro. In neuron cells, p5 inhibited deregulated Cdk5-p25 activity but had no effect on the activity of endogenous Cdk5-p35 or on any related endogenous cyclin-dependent kinases in HEK293 cells. Specificity of p5 inhibition in cortical neurons may depend on the p10 domain in p35, which is absent in p25. Furthermore, we have demonstrated that p5 reduced Aβ(1-42)-induced Tau hyperphosphorylation and apoptosis in cortical neurons. These results suggest that p5 peptide may be a unique and useful candidate for therapeutic studies of certain neurodegenerative diseases.

Noninvasive Probing of Inhibitory Effects of Cylindrospermopsin and Microcystin-LR Using Cell-Based Impedance Spectroscopy

In an effort to develop a noninvasive method for assessment of cyanobacterial toxins in drinking water, plausible cytotoxicity/inhibition of microcystin-LR and cylindrospermopsin was evaluated by cell-substrate impedance sensing (ECIS) using three different cell lines. Sf9 insect cells were attached to concanavalin A coated gold electrodes, whereas Chinese hamster ovary (CHO) and human embryo kidney (HEK) cells were attached to a fibronectin or laminin coated gold surface. Cytotoxic or inhibitory effects were dependent upon the cell line and the extracellular matrix (ECM) coating. Neither toxin exhibited any appreciable effect on the insect cells. In contrast, cytotoxicity of cylindrospermopsin on CHO cells was attested by both ECIS and viability tests. The half-inhibition concentration (ECIS50) of cylindrospermopsin for CHO cells was approximately 2 microg/mL (ppm) after 20 h of exposure and 4 microg/mL (ppm) after 30 h of exposure for a laminin or fibronectin coated surface. ECIS confirmed no significant effect of cylindrospermopsin on HEK cells. Microcystin-LR was also tested with CHO cells, resulting in an ECIS50 value of approximately 12 microg/mL (ppm) after 25 h of exposure for a laminin coated gold surface. The effect of microcystin-LR on CHO cells probed by ECIS was inhibitory rather than cytotoxic, as confirmed by cell viability assays.

In vitro inhibition of CSFV replication by retroviral vector-mediated RNA interference

Classical swine fever (CSF) is a highly contagious viral disease of pigs which causes major economic losses worldwide. No specific drug is currently available for the effective treatment of CSFV infection. RNA interference (RNAi) technology depends on effective delivery systems, for which several effective vectors have recently been developed. Three retroviral plasmids containing siRNA genes targeting different regions of N pro and NS4A have been constructed, and 3 replication-incompetent retroviral vectors have been produced in the human embryo kidney cell line GP2-293 by retroviral plasmid transfection. PK-15 cells were then infected with these replication-incompetent retroviral vectors and screened for siRNA stably expressing PK-15 cell clones. Growth of CSFV in such siRNA stably expressing cell clones resulted in a 186-fold reduction in viral genome copies and, at 72 h post-infection, only a small % of cells showed infection by indirect immunofluorescence microscopy, and effective inhibition of virus replication persisted for up to 120 h. Retroviral vector-mediated RNAi can therefore be used to study the specific function of viral genes associated with CSFV replication and may have potential therapeutic application.

Interaction between nuclear localization signal-retinoic acid receptor alpha and Ubiquilin 1.

To identify the interaction between nuclear localization signal-retinoic acid receptor alpha (NLS-RARalpha) and Ubiquilin 1(UBQLN1). The recombination expression plasmids pGBKT7-NLS-RAR and pACT2-UBQLN1, which expressed bait-protein NLS-RARalpha and target protein UBQLN1 respectively, were cotransformated into yeast AH109. The interaction of the expression plasmids in the living cells was investigated by yeast two-hybrid assay. HA-tagged fusion protein (pCMV-HA-NLS-RARalpha) expression vector and Myc-tagged fusion protein (pCMV-Myc-JTV1) expression vector were constructed, identified, and cotransfected respectively into human embryo kidney 293 cells(HEK293). The interaction was detected by co-immunoprecipitation. Blue clones were found on yeast AH109 plate cotransformated with pGBKT7-NLS-RARalpha and pACT2-UBQLN1. Double restriction enzyme digestion showed that pCMV-HA-NLS-RARalpha and pCMV-Myc-JTV1 were successfully constructed. Then HA-NLS-RAR protein was immunoprecipitated by anti-HA polyclonal antibody and the expression of Myc-UBQLN1 was tested by Western blot with anti-Myc monoclonal antibody from immunoprecipitated complex. The interaction between NLS-RARalpha and UBQLN1 can be verified by yeast two-hybrid assay and co-immunoprecipitation.

2,2′,4′‐Trihydroxychalcone from Glycyrrhiza glabra as a new specific BACE1 inhibitor efficiently ameliorates memory impairment in mice

Alzheimer's disease (AD) characterizes a progressive neurodegenerative disorder of the brain, while AD patients are afflicted with irreversible loss of neurons and further the intellectual abilities including memory and reasoning. One of the typical hallmarks of AD is the deposition of senile plaque that is contributed mainly by amyloid-beta (Abeta), whose production is initiated by beta-site amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1). Inhibition of BACE1 is thereby regarded as an attractive strategy for anti-AD drug discovery. Here, we reported that the natural product 2,2',4'-trihydroxychalcone (TDC) from Glycyrrhiza glabra functioned as a specific non-competitive inhibitor against BACE1 enzyme, and potently repressed beta-cleavage of APP and production of Abeta in human embryo kidney cells-APPswe cells. Moreover, the amelioration ability of this compound against the in vivo memory impairment was further evaluated by APP-PS1 double transgenic mice model. It is discovered that treatment of 9 mg/kg/day of TDC could obviously decrease Abeta production and Abeta plaque formation, while efficiently improve the memory impairment based on Morris water maze test. Our findings thus demonstrated that the natural product TDC as a new BACE1 inhibitor could ameliorate memory impairment in mice, and is expected to be potentially used as a lead compound for further anti-AD reagent development.

Y65C Missense Mutation in the WW Domain of the Golabi-Ito-Hall Syndrome Protein PQBP1 Affects Its Binding Activity and Deregulates Pre-mRNA Splicing

The PQBP1 (polyglutamine tract-binding protein 1) gene encodes a nuclear protein that regulates pre-mRNA splicing and transcription. Mutations in the PQBP1 gene were reported in several X chromosome-linked mental retardation disorders including Golabi-Ito-Hall syndrome. The missense mutation that causes this syndrome is unique among other PQBP1 mutations reported to date because it maps within a functional domain of PQBP1, known as the WW domain. The mutation substitutes tyrosine 65 with cysteine and is located within the conserved core of aromatic amino acids of the domain. We show here that the binding property of the Y65C-mutated WW domain and the full-length mutant protein toward its cognate proline-rich ligands was diminished. Furthermore, in Golabi-Ito-Hall-derived lymphoblasts we showed that the complex between PQBP1-Y65C and WBP11 (WW domain-binding protein 11) splicing factor was compromised. In these cells a substantial decrease in pre-mRNA splicing efficiency was detected. Our study points to the critical role of the WW domain in the function of the PQBP1 protein and provides an insight into the molecular mechanism that underlies the X chromosome-linked mental retardation entities classified globally as Renpenning syndrome.

Characterization of the Substrate Specificity of Human Carboxypeptidase A4 and Implications for a Role in Extracellular Peptide Processing

CPA4 (carboxypeptidase A4) is a member of the metallocarboxypeptidase family. CPA4 was originally found in a screen of mRNAs up-regulated by sodium butyrate-induced differentiation of cancer cells. Further studies suggested a relation between CPA4 and prostate cancer aggressiveness. In the present study, we determined that CPA4 is secreted from cells as a soluble proenzyme (pro-CPA4) that can be activated by endoproteases, such as trypsin. Three complementary approaches were used to study the substrate specificity of CPA4; kinetic analysis was performed using a new series of chromogenic substrates and some biologically relevant peptides, the cleavage of synthetic peptides was tested individually, and the cleavage of a mixture of >100 mouse brain peptides was examined using a quantitative peptidomics mass spectrometry-based approach. CPA4 was able to cleave hydrophobic C-terminal residues with a preference for Phe, Leu, Ile, Met, Tyr, and Val. However, not all peptides with C-terminal hydrophobic residues were cleaved, indicating the importance of additional residues within the peptide. Aliphatic, aromatic, and basic residues in the P1 position have a positive influence on the cleavage specificity. In contrast, acidic residues, Pro, and Gly have a negative influence in the P1 position. Some of the peptides identified as CPA4 substrates (such as neurotensin, granins, and opioid peptides) have been previously shown to function in cell proliferation and differentiation, potentially explaining the link between CPA4 and cancer aggressiveness. Taken together, these studies suggest that CPA4 functions in neuropeptide processing and regulation in the extracellular environment.

The influence of over expression of interleukin-1 receptor-associated kinase 1 on bacterial lipoprotein-induced tolerance

To investigate Toll-like receptor 2 (TLR2) and interleukin-1 receptor-associated kinase 1 (IRAK-1) in bacterial lipoprotein (BLP) tolerance. Western blotting was used to confirm the over expression of TLR2 and IRAK-1 in human embryo kidney 293 (HEK293) cells. Plasmids for dual luciferase reporter gene with nuclear factor-KappaB promoter (pNF-KappaB-Luc) or CMV promoter (phRL-CMV internal control vector) were used to detect the NF-KappaB activation and the induction of BLP tolerance in HEK-TLR2 cells. BLP stimulation resulted in dose-dependent NF-KappaB activation in HEK293 cells stably expressing TLR2. And BLP pretreatment could reduce NF-KappaB activation and induce BLP tolerance in HEK-TLR2 cells. The NF-KappaB activation was 0.329+/-0.010 and 0.168+/-0.010 in BLP-activated and BLP-tolerant HEK-TLR2 cells, respectively. After transfection with 0.02 microg IRAK-1 plasmid, NF-KappaB activation in the two groups was 0.493+/-0.010 and 0.427+/-0.035, respectively (both P<0.01). So over expression of IRAK-1 could increase NF-KappaB activation in a dose-dependent manner. These results demonstrated that over expression of IRAK-1 could reverse BLP tolerance, whereas over expression of TLR2 failed to prevent the induction of BLP tolerance. Therefore reduced IRAK-1 protein expression is an important mechanism in the development of BLP-induced tolerance, suggesting that it could be a potentially important target for future therapeutic strategies in bacterial infection and sepsis.

Construction and characterization of calreticulin-HBsAg fusion gene recombinant adenovirus expression vector

To generate recombinant adenoviral vector containing calreticulin (CRT)-hepatitis B surface antigen (HBsAg) fusion gene for developing a safe, effective and HBsAg-specific therapeutic vaccine. CRT and HBsAg gene were fused using polymerase chain reaction (PCR), endonuclease digestion and ligation methods. The fusion gene was cloned into pENTR/D-TOPO transfer vector after the base pairs of DNA (CACC) sequence was added to the 5' end. Adenoviral expression vector containing CRT-HBsAg fusion gene was constructed by homologous recombinantion. The human embryo kidney (HEK) 293A cells were transfected with linearized DNA plasmid of the recombinant adenoviral vector to package and amplify recombinant adenovirus. The recombinant adenovirus titer was characterized using the end-dilution assay. The expression of the CRT/HBsAg fusion protein in Ad-CRT/HBsAg infected 293A cells was detected by Western blotting. The CRT-HBsAg fusion gene was characterized by PCR and sequencing and its length and sequence were confirmed to be accurate. The CRT-HBsAg fusion gene recombinant pENTR/D-TOPO transfer vector was constructed. The recombinant adenoviral vector, Ad-CRT/HBsAg, was generated successfully. The titer of Ad-CRT/HBsAg was characterized as 3.9 x 10(11) pfu/mL. The CRT-HBsAg fusion protein was expressed by HEK 293A cells correctly. CRT/HBsAg fusion gene recombinant replication-defective adenovirus expression vector is constructed successfully and this study has provided an experimental basis for further studies of Hepatitis B virus gene therapy.

Real Time Detection of Mechanical Stress in Specific Cytoskeletal Proteins

A molecular force sensor cassette (stFRET) was incorporated into actinin, filamin, and spectrin in endothelial cells and into collagen-19 in C. elegans. In vitro double strand DNA stretching essay confirmed the force sensitivity of stFRET. stFRET detected constitutive stress in all the proteins. In endothelial cells, the stress in actinin, filamin and spectrin could be eliminated by releasing focal attachments from the substrate. Our data also indicated the highest resting strain in spectrin in three cytoskeleton protein tested. When the intact C. elegans worm was reversibly stretched using micromanipulators, stFRET reversibly sensed the force in collagen-19. stFRET is a general purpose dynamic sensor of mechanical stress in filamentous proteins that can be expressed in cells in vitro and in whole animals.We have developed another force sensor, named sstFRET, in which we substituted the alpha helix linker with a spectrin repeat domain. Our in vitro DNA stretching assay showed that 20 pN force is sufficient to deform the spectrin repeat linker, leading to a dramatic change of FRET. By inserting sstFRET into alpha-actinin, we succeeded in monitoring the change in actinin constitutive stress before and after cells are osmotically challenged. In addition, we observe substantially lower force loads on actinin in bovine aortic endothelial cells (BAEC) than that in human embryo kidney (HEK) cells.

High-Photoluminescence-Yield Gold Nanocubes: For Cell Imaging and Photothermal Therapy

Gold nanocubes demonstrate unique optical properties of the high photoluminescence (PL) quantum yield and a remarkably enhanced extinction band at 544 nm. The 4 x 10(-2) PL yield, which is about 200 times higher than that of gold nanorods, allows gold nanocubes to be successfully used in cell imaging of human liver cancer cells (QGY) and human embryo kidney cells (293T) with a common method of single-photon excitation. The high extinction coefficients of gold nanocubes also facilitate them carrying out the photothermal therapy of QGY and 293T cells, showing similar photokilling efficiency as compared to gold nanorods.

The unique role of proprotein convertase subtilisin/kexin 9 in cholesterol homeostasis

The LDL receptor (LDLR) plays an essential role in the regulation of plasma (LDL) cholesterol concentrations by virtue of its ability to clear plasma LDL. Down-regulation of the LDLR by proprotein convertase subtilisin/kexin 9 (PCSK9) has recently emerged as a regulatory mechanism that controls plasma LDL cholesterol concentrations. Studies in which PCSK9 is over-expressed in mice, have demonstrated that PCSK9, by enhancing hepatic LDLR degradation, decreases the availability of the LDLR for LDL uptake, resulting in increased plasma LDL cholesterol levels. However, PCSK9 has also recently been shown to mediate down-regulation of surface receptors other than the LDLR, suggesting that it may have much broader roles than initially thought.

Phagosomal retention of Francisella tularensis results in TIRAP/Mal-independent TLR2 signaling

TLR2 plays a central role in the activation of innate immunity in response to Ft, the causative agent of tularemia. We reported previously that Ft LVS elicited strong, dose-dependent NF-kappaB reporter activity in TLR2-expressing human embryo kidney 293 T cells and that Ft LVS-induced murine macrophage proinflammatory cytokine gene and protein expression is TLR2-dependent. We demonstrated further that Ft can signal through TLR2 from within the phagosome and that phagosomal retention of Ft leads to greatly increased expression of a subset of proinflammatory genes. The two adaptor proteins associated with TLR2-mediated signaling are MyD88 and TIRAP. Although MyD88 is absolutely required for the Ft-induced macrophage cytokine response, the requirement for TIRAP can be overcome through retention of Ft within the phagosome. TIRAP-independent signaling was observed whether Ft was retained in the phagosome as a result of bacterial mutation (LVSDeltaiglC) or BFA-mediated inhibition of phagosome acidification. The requirement for TIRAP in TLR2 signaling could also be overcome by increasing the concentrations of synthetic bacterial TLR2 agonists. Taken together, these data suggest that prolonging or enhancing the interaction between TLR2 and its agonist overcomes the "bridging" function ascribed previously to TIRAP.

더덕 추출물의 항돌연변이 및 항종양 효과

This study was carried out to investigate the mutagenic, antimutagenic, cytotoxicity and antitumor effect of Codonopsis lanceolata (CL). CL was extracted with 70% ethanol and then further fractionated to hexane, chloroform, ethyl acetate, butanol and water. Antimutagenic, cytotoxicity and antitumor effects of CL extracts were measured by using Ames test, SRB method, and the tumor growth inhibition test. CL extracts did not show any mutagenicity in the Ames test; however, 70% ethanol extracts and its fractions had strong antimutagenic effects against mutation induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and 4-nitroquinoline-1-oxide (4NQO). The ethyl acetate fraction of CL (200 ㎍/plate) showed approximately 72.1% inhibitory effect on the mutagenesis induced by 4NQO against TA98 strain, whereas 69.6% and 67.0% inhibitions were observed on the mutagenesis induced by MNNG and 4NQO against TA100 strain. In anticancer effects, the cytotoxicity of CL extract and its fractions against cancer cell lines including human cervical adenocarcinoma (HeLa), human hepatocellular carcinoma (HepG2), human breast adenocarcinoma (MCF-7), human lung carcinoma (A549) and transformed primary human embryo kidney (293) were investigated. The treatment of 1 ㎎/mL CL ethyl acetate fraction had the highest cytotoxicity of 74.5%, 70.7% and 80.3% against HeLa, MCF-7 and A549 cells, respectively. In contrast, the extract and its fractions showed only 2∼31% cytotoxicity for a normal human kidney cell line (293). In vivo anticancer effect of CL extract was tested using Balb/c mice transplanted sarcoma-180 cells. CL ethyl acetate fraction showed the highest inhibition rate of 56.4% at the 50 ㎎/㎏ concentration.

Synthesis of 2‐Fluoro N10‐Substituted Acridones and Their Cytotoxicity Studies in Sensitive and Resistant Cancer Cell Lines and Their DNA Intercalation Studies

A series of 2-fluoro N(10)-substituted acridone derivatives with varying alkyl side chain length with propyl, butyl substitution, and a tertiary amine group at the terminal end of the alkyl side chain were synthesized and screened against cancer cell lines SW 1573, SW 1573 2R 160 (P-gp substrate) which are non-small lung cancer cell lines, MCF-7, MCF-7/MR (BCRP substrate) are breast cancer cell lines, 2008 WT, 2008MRP1, 2008MRP2, 2008MRP3 are ovarian cancer cell lines, and human embryo kidney cell lines like HEK293, HEK293 MRP4, and HEK293 MRP5i. The propyl-series compounds showed lipophilicity in the range of 1.93 to 4.40 and the butyl series in the range of 2.37 to 4.78. The compounds 4, 7, and 8 showed good cytotoxicity against the 60 human cancer cell line panel of the National Cancer Institute, USA. The compounds 14 and 15 showed a better cytotoxicity in most of the cancer cell lines compared to other compounds tested. The DNA-binding properties of the compounds were evaluated based on their affinity or intercalation with CT-DNA measured with absorption titration. The compound 11 bearing planar tricyclic ring linked with a butyl methylpiperazino side chain showed the highest binding affinity with a binding constant (K(i)) of 10.38 x 10 M(-1). Evaluation of the compounds in cell lines with an overexpression of various multidrug resistance-related protein (MRP), P-glycoprotein (P-gp), or Breast Cancer Resistance Protein (BCRP) showed that all compounds are not substrates for any of these transporters.

Cloning and characterization of a novel intracellular protein p48.2 that negatively regulates cell cycle progression

Neurofibromatosis type 1 (NF1) microdeletion is a large genomic deletion that embraces at least 11 continuous genes at human chromosome 17q11.2. To date, most of these genes’ functions still remain undefined. In this study, we report an unknown cytokine receptor like molecule (p48.2) that is frequently deleted in patients with type-1 and type-2 NF1 microdeletions in the neurofibromin locus. The cloned gene has 1317 base pair long that encodes a 438aa intracellular protein. The gene was subsequently named p48.2 based on its predicted molecular weight. A typical fibronectin type III (FNIII) domain was identified in p48.2 between Arg176 and Pro261 in which a palindromic Arg-Gly-Asp (RGD) repeat plus a putative Trp-Ser-X-Trp-Ser (WSXWS) motif were found at the domain's C-terminus. p48.2 mRNAs were abundant in many tumor cell lines and normal human tissues and up-regulated in some freshly isolated lung cancer and leukemia cells. Interestingly, over-expression of p48.2 in human embryo kidney 293T cells could significantly cause G0/G1 arrest and prevented S phase entry. In contrast, repressing endogenous p48.2 gene expression by specific siRNA markedly reduced G0/G1 population. Importantly, over-expression of p48.2 could significantly up-regulate rather than down-regulate cyclin D1 and cyclin D3 expressions. We further showed that the induction of cyclin D1 expression was directly due to the activation of signal transducers and activators of transcription 3 (STAT3), but was independent of RAS/mitogen-activated protein kinase (RAS/MAPK) signaling pathway. Thus, p48.2 may represent a novel type of intracellular protein functioning as a negative regulator at the G0/G1 phase.

A robust method for the joint estimation of yield coefficients and kinetic parameters in bioprocess models

Bioprocess model structures that require nonlinear parameter estimation, thus initialization values, are often subject to poor identification performances because of the uncertainty on those initialization values. Under some conditions on the model structure, it is possible to partially circumvent this problem by an appropriate decoupling of the linear part of the model from the nonlinear part of it. This article provides a procedure to be followed when these structural conditions are not satisfied. An original method for decoupling two sets of parameters, namely, kinetic parameters from maximum growth, production, decay rates, and yield coefficients, is presented. It exhibits the advantage of requiring only initialization of the first subset of parameters. In comparison with a classical nonlinear estimation procedure, in which all the parameters are freed, results show enhanced robustness of model identification with regard to parameter initialization errors. This is illustrated by means of three simulation case studies: a fed-batch Human Embryo Kidney cell cultivation process using a macroscopic reaction scheme description, a process of cyclodextrin-glucanotransferase production by Bacillus circulans, and a process of simultaneous starch saccharification and glucose fermentation to lactic acid by Lactobacillus delbrückii, both based on a Luedeking-Piret model structure. Additionally, perspectives of the presented procedure in the context of systematic bioprocess modeling are promising.

C-Abl Phosphorylates Hdmx and Regulates Its Interaction with p53

Upon exposure to DNA damage the p53 tumor suppressor is accumulated and activated to stall cellular growth. For this to occur, p53 must be relieved from its major inhibitors, Mdm2 (Hdm2 in humans) and Mdmx (Mdm4; Hdmx in humans). A key mechanism controlling this relief is the post-translational modifications of p53 and its inhibitors. We have previously demonstrated that the stress-activated tyrosine kinase, c-Abl, contributes to the relief of p53 from Hdm2. Because Hdmx is the major inhibitor of p53 activity, the additional possibility that c-Abl protects p53 through targeting Hdmx was explored in this study. c-Abl was found to interact with and to phosphorylate Hdmx. This phosphorylation was enhanced in response to DNA damage. Importantly, we mapped the sites of phosphorylation to the p53 binding domain of Hdmx. One of these phosphorylations, on tyrosine 99, inhibited Hdmx interaction with p53. This inhibition is consistent with the predicted role of this residue in the interaction with p53 based on the crystal structure of the interaction site. Our results show that c-Abl not only targets Hdm2, but also Hdmx, which together contribute to p53 activation in response to DNA damage.