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When entering circulation, multi-walled carbon nanotubes (MWCNTs) will inevitably adsorb proteins, which can consequently influence their toxicity to cells lining human blood vessels. In this study, we investigated the influence of pre-incubation with bovine serum albumin (BSA) on internalization, cytotoxicity, oxidative stress and inflammation induced by pristine/carboxylated MWCNTs to human umbilical vein endothelial cells (HUVECs). Atomic force microscopy (AFM) indicated the adsorption of proteins onto the surface of MWCNTs, which consequently increased the diameter. Pre-incubation with BSA did not obviously influence the hydrodynamic sizes, but decreased the zeta potential of MWCNTs. Transmission electron microscopy (TEM) indicated the internalization of both types of MWCNTs into HUVECs, whereas pre-incubation with BSA appeared to enhance the internalization. MWCNT exposure induced cytotoxicity and oxidative stress, as well as a modest inflammatory response shown as an increased THP-1 adhesion to HUVECs, but not release of interleukin 6 (IL-6) or tumor necrosis factor (TNFα). Exposure to MWCNTs pre-incubated with BSA induced less cytotoxicity to HUVECs, associated with increased intracellular glutathione (GSH). However, MWCNTs induced IL-6 and TNFα release, as well as THP-1 adhesion to HUVECs, were enhanced after pre-incubation with BSA. In summary, these data indicated that pre-incubation with BSA could enhance the internalization of MWCNTs to HUVECs, which consequently reduces the cytotoxicity and oxidative stress, but enhances the inflammatory response of MWCNTs. The reduced cytotoxicity and oxidative stress, and enhanced inflammatory responses are likely due to a combined effect of BSA and MWCNTs, which suggests that when assessing the biological effects of MWCNTs in circulation, it is necessary to consider the interactions between MWCNTs and serum proteins.

Two novel thiadiazolo[3,4-g]quinoxaline (TQ) derivatives, 6,7-bis(4-(hexyloxy)phenyl)-[1,2,5]thiadiazolo[3,4- g]quinoxaline (TQs-1) and 4,9-dibromo-6,7-bis(4-(hexyloxy)phenyl)-[1,2,5]thiadiazolo[3,4-g]quinoxaline (TQs-2) were synthesized and fully characterized by 1H-NMR, 13C-NMR and HR-MS spectra. Their photo-physical and photo-chemical properties, including UV-Vis spectra, fluorescence spectra, photostability and singlet oxygen quantum yield were comprehensively studied. Compared to TQs-1, TQs-2 exhibited a higher singlet oxygen quantum yield and a broader absorption spectrum due to the introduction of two bromine atoms. Furthermore, TQs-2 based nanoparticles (TQs-2 NPs) was obtained via a simple co-assembly strategy using a biocompatible amphiphilic polymer DSPE-mPEG2000 to encapsulate TQs-2. In vitro photodynamic therapy (PDT) experiments to three tumor cells (4T1, MCF-7 and HeLa) and normal cells (L929) were carried out using TQs-2 NPs as photosensitizers. The results showed that TQs-2 NPs possessed negligible dark cytotoxicity to these cells at a high concentration of 50 μg mL-1, but exhibited high light cytotoxicity to them, especially to three tumor cells, under a 532 nm laser illumination at the same concentration, indicating that TQs-2 NPs has a promising potential for PDT applications.

Two novel thiadiazolo[3,4-g]quinoxaline (TQ) photosensitizers (PSs), TQs-3 and TQs-4, were designed and synthesized. Both of them presented ultra-high singlet oxygen quantum yields under red light irradiation. By carrying out in vitro photodynamic therapy (PDT) experiments using TQs-4 loaded nanoparticles (TQs-4 NPs) to treat three kinds of tumor cell lines: 4T1, HeLa and MCF-7 cells, it was demonstrated that TQs-4 NPs had outstanding PDT efficacies. An ultra-low dose of TQs-4 (0.14 μg mL-1) can realize the death of more than 90% HeLa cells (635 nm, 60 mW cm-2, 10 min), which indicated that TQs-4 show promising potential as a novel PS for PDT applications.

Background Melophagus ovinus (sheep ked) is a blood-feeding ectoparasite that belongs to the family Hippoboscidae (Diptera: Hippoboscoidea) and mainly parasitizes sheep. The life-cycle of M. ovinus consists of three stages: larva, pupa and adult. It has a worldwide distribution and has been found in four provinces of China, especially South Xinjiang. In addition to causing direct damage to animal hosts, M. ovinus serves as a vector for disease transmission. In this study, our aim was to investigate the presence of Anaplasma spp. in pupal and adult M. ovinus. Methods A total of 93 specimens (including eight pupal specimens) of M. ovinus collected in South Xinjiang were selected for isolation of genomic DNA, followed by PCR amplification and sequencing of the msp4 gene of Anaplasma spp. The sequences were analyzed in MEGA 7.0 software and via online BLAST. Results PCR and sequencing results showed that all the specimens collected in 2013 were free of Anaplasma spp., whereas three and 25 specimens (including five pupal specimens) collected in 2016 and 2017, respectively, tested positive for Anaplasma spp. The analysis of 24 msp4 gene sequences (from four pupal specimens) confirmed the presence of A. ovis in M. ovinus specimens collected in South Xinjiang, China. The detected A. ovis isolates belong to Genotypes II and III. Conclusions To the best of our knowledge, this is the first report of the detection of A. ovis DNA in pupal M. ovinus, confirming the vertical transmission of A. ovis in M. ovinus and the potential of M. ovinus to serve as a vector for A. ovis.

It is recently shown that biological macromolecules in food could interact with nanoparticles (NPs) and consequently change the biological effects of NPs. In this study, the interactions between ZnO NPs with or without hydrophobic surface coating and bovine serum albumin (BSA) or oleate (OA) complexed to BSA (OA-BSA) were assessed. Atomic force microscope (AFM) showed topographic changes of both types of NPs by BSA or OA-BSA, which could indicate the formation of protein corona. ZnO NPs showed negative Zeta potential, which was slightly decreased by BSA or OA-BSA, with OA-BSA being more effective. The UV–Vis was increased, whereas the fluorescence and synchronous fluorescence was decreased by the presence of ZnO NPs. Exposure to both types of ZnO NPs was associated with cytotoxicity to THP-1 macrophages, which was equally mitigated by BSA or OA-BSA associated with decreased cellular Zn elements. Exposure to ZnO NPs was associated with decreased release of cytokines, which was not affected by BSA or OA-BSA. In combination, the results from this study suggested that both BSA and OA-BSA could be adsorbed to ZnO NPs regardless of hydrophobic surface coating, which reduced the cytotoxicity of NPs to macrophages probably due to reduced association between NPs and cells. BSA and OA-BSA equally protected THP-1 macrophages from ZnO NP exposure, which might indicate that complexation to OA did not compromise the cytoprotective effects of BSA. These data might also indicate the complex interaction between NPs and biological macromolecules as food components, which should be considered for future nanotoxicological studies.

Background The endoplasmic-reticulum (ER) responds to the burden of unfolded proteins in its lumen by activating intracellular signal transduction pathways, also known as the unfolded protein response (UPR). Many signal transduction events and transcription factors have been demonstrated to be associated with ER stress. The process in which ER stress affects or interacts with other pathways is still a progressing topic that is not completely understood. Identifying new transcription factors associated with ER stress pathways provides a platform to comprehensively characterize mechanism and functionality of ER. Methods We utilized a transcription factor (TF) activation plate array to profile the TF activities which were affected by ER stress induced by pharmacological agents, thapsigargin (TG) and tunicamycin (TM) at 1 h, 4 h, 8 h and 16 h respectively, in MiaPACA2 cells. The altered activity patterns were analyzed and validated using gel shift assays and cell-based luciferase reporter assay. Results The study has not only confirmed previous findings, which the TFs including ATF4, ATF6, XBP, NFkB, CHOP and AP1, were activated by ER stress, but also found four newly discovered TFs, NFAT, TCF/LEF were activated, and PXR was repressed in response of ER stress. Different patterns of TF activities in MiaPaCa2 were demonstrated upon TM or TG treatment in the time course experiments. The altered activities of TFs were confirmed using gel shift assays and luciferase reporter vectors. Conclusion This study utilized a TF activation array technology to identify four new TFs, HIF, NFAT, TCF/LEF and PXR that were changed in their activity as a result of ER stress induced by TG and TM. The TF activity patterns were demonstrated to be diverse in response to the duration of TG or TM treatment. These new findings will facilitate further unveiling the complex mechanisms of the ER stress process and associated diseases.

Using luciferase reporter constructs driven by specific promoter response elements, we developed a series of stable reporter cell lines for monitoring the activity of specific transcription factors (TFs). These TFs, which play essential roles in regulating diverse biological functions, include nuclear factor kappaB (NFkappaB), cyclic AMP response element-binding protein, activator protein 1, signal transducer and activator of transcription 1 and 3, nuclear factor of activated T cells, serum response factor, and hypoxia-inducible factor. The response of the stable reporter cells was highly specific. For example, tumor necrosis factor-alpha (TNFalpha) strongly activated NFkappaB reporter cells, but not other cell lines. The NFkappaB reporter was active in multiple cell lines, including 293T, HeLa, A549, and NIH3T3 cells, in response to TNFalpha, indicating that this system is useful to monitor specific TFs in different model cell lines. To facilitate high throughput screening of these cell lines, they were adapted to a 96-well format. These stable reporter cells are also applicable for the analysis of steroid hormone receptors, which bind directly to the response element after ligand binding. With the HeLa/glucocorticoid response element-luciferase stable reporter cells, we were able to discriminate pharmacological activity of different compounds for the glucocorticoid receptor. Taken together, these results demonstrate that the stable reporter cells are useful tools for: (1) detection of signaling pathway-specific ligands; (2) identification of novel ligands for specific TFs, and (3) screening for agonists and antagonists of specific ligands/receptors.

Transcription factors (TFs), which play crucial roles in the regulation of gene expression in the human genome, are highly regulated by a variety of mechanisms. A single extracellular stimulus can trigger multiple signaling pathways, and these in turn can activate multiple TFs to mediate the inducible expression of target genes. Alterations in the activities of TFs are often associated with human diseases, such as altered activating factor 1, estrogen receptor, and p53 function in cancer, nuclear factor kappaB in inflammatory diseases, and peroxisome proliferator-activated receptor gamma in obesity. A systematic assay for profiling the activation of TFs will aid in elucidating the mechanisms of TF activation, reveal altered TFs associated with human diseases, and aid in developing assays for drug discovery. Here, we developed a 24-plex fluorescent microsphere-based TF activation assay system with a 96-well plate format. The assay system enabled high-throughput profiling of the DNA binding activity of TFs in multiple samples with high sensitivity.

Src homology 2 (SH2) domains are evolutionary conserved small protein modules that bind specifically to tyrosine-phosphorylated peptides. More than 100 SH2 domains have been identified in proteins encoded by the human genome. The binding specificity of these domains plays a critical role in signaling within the cell, mediating the relocalization and interaction of proteins in response to changes in tyrosine phosphorylation states. Here we developed an SH2 domain profiling method based on a multiplexed fluorescent microsphere assay in which various SH2 domains are used to probe the global state of tyrosine phosphorylation within a cell and to screen synthetic peptides that specifically bind to each SH2 domain. The multiplexed, fluorescent microsphere-based assay is a recently developed technology that can potentially detect a wide variety of interactions between biological molecules. We constructed 25-plex SH2 domain-GST fusion protein-conjugated fluorescent microsphere sets to investigate phosphorylation-mediated cell signaling through the specific binding of SH2 domains to activated target proteins. The response of HeLa, COS-1, A431, and 293 cells and four breast cancer cell lines to epidermal growth factor and insulin were quantitatively profiled using this novel microsphere-based, multiplexed, high throughput assay system.

Multiple signal transduction pathways are generally triggered simultaneously by a single extracellular stimulus. As a result, multiple transcription factors (TFs) can be activated downstream to mediate the inducible expression of target genes. Profiling the activation of all TFs will aid in the dissection of the numerous pathways of signal transduction. Tumor necrosis factor alpha (TNFalpha) and phorbol 12-myristate 13-acetate (PMA) mediate many biological functions, including cell proliferation and apoptosis, by stimulating signaling pathways. Two TFs, nuclear factor kappaB (NFkappaB) and activating factor 1 (AP1), have been identified as targets of both TNFalpha and PMA activation. Here, we describe the use of a protein/DNA array system to identify additional TFs activated by TNFalpha and PMA in HeLa cells. From a total of 150 targeted TFs, six-CREB, E2F, CETP/CRE, c-Rel, MSP1, and Pax6-were identified whose activities, like NFkappaB and AP1, were regulated by both TNFalpha- and PMA-induced pathways. Interestingly, the TF E47 was shown to be specifically activated by TNFalpha but was not affected by treatment with PMA. In addition, GATA, NF-E1, and ISRE were shown to be specifically activated by PMA but not TNFalpha. These findings suggest that TNFalpha and PMA both stimulate unique signaling pathways while mediating transcriptional activation through common pathways.

A comparative in vivo pharmacokinetic (PK) study of tilmicosin (TIL) was conducted in 6 crossbred healthy pigs and 6 crossbred pigs infected with Haemophilus ( H .) parasuis following oral administration of a single 40 mg/kg dose. The infected model was established by intranasal inoculation and confirmed by clinical signs, blood biochemistry, and microscopic examinations. Plasma TIL concentrations were determined by a validated high-performance liquid chromatography method with ultraviolet detection at 285 nm. PK parameters were calculated by using WinNonlin software. After TIL administration, the main PK parameters of TIL in healthy and H. parasuis -infected pigs were as follows: Area under the concentration-time curve, maximal drug concentration, half-life of the absorption phase, half-life of the distribution phase, and half-life of the elimination phase were 34.86 ± 9.69 vs. 28.73 ± 6.18 μgㆍh/mL, 1.77 ± 0.33 vs. 1.67 ± 0.28 μg/mL, 2.27 ± 0.45 vs. 2.24 ± 0.44 h, 5.35 ± 1.40 vs. 4.61 ± 0.35 h, and 43.53 ± 8.17 vs. 42.05 ± 9.36 h, respectively. These results of this exploratory study suggest that there were no significant differences between the PK profiles of TIL in the healthy and H. parasuis -infected pigs.

The green fluorescent protein (GFP) is a widely used reporter in gene expression and protein localization studies. GFP is a stable protein; this property allows its accumulation and easy detection in cells. However, this stability also limits its application in studies that require rapid reporter turnover. We created a destabilized GFP for use in such studies by fusing amino acids 422-461 of the degradation domain of mouse ornithine decarboxylase (MODC) to the C-terminal end of an enhanced variant of GFP (EGFP). The fusion protein, unlike EGFP, was unstable in the presence of cycloheximide and had a fluorescence half-life of 2 h. Western blot analysis indicated that the fluorescence decay of EGFP-MODC-(422-461) was correlated with degradation of the fusion protein. We mutated key amino acids in the PEST sequence of EGFP-MODC-(422-461) and identified several mutants with variable half-lives. The suitability of destabilized EGFP as a transcription reporter was tested by linking it to NFkappaB binding sequences and monitoring tumor necrosis factor alpha-mediated NFkappaB activation. We obtained time course induction and dose response kinetics similar to secreted alkaline phosphatase obtained in transfected cells. This result did not occur when unmodified EGFP was used as the reporter. Because of its autofluorescence, destabilized EGFP can be used to directly correlate gene induction with biochemical change, such as NFkappaB translocation to the nucleus.

The Green Fluorescent Protein (GFP) from the jellyfish Aequorea victoria is a widely used marker for gene expression and protein localization studies. Dissection of the structure of the protein would be expected to shed light on its potential applications to other fields such as the detection of protease activity. Using deletion analysis, we have defined the minimal domain in GFP required for fluorescence to amino acids 7-229. This domain starts at the middle of the first small alpha helix at the N terminus of GFP and ends immediately following the last beta sheet. Studies of the amino acids at both termini of the minimal domain revealed that positions 6 and 7 at the N terminus are Glu-specific. Change of the Glu residues to other amino acids results in reduction of GFP fluorescence. Position 229 at the C terminus of GFP, however, is nonspecific: the Ile can be replaced with other amino acids with no measurable loss of fluorescence. A total of only 15 terminal amino acids can be deleted from GFP without disrupting fluorescence, consistent with findings of a previous study of GFP crystal structure (Ormo, M., Cubitt, A. B., Kallio, K., Gross, L. A., Tsien, R. Y., Remington, S. J. (1996) Science 273, 1392-1395 and Yang, F., Moss, L. G., and Phillips, G. N., Jr. (1996) Nat. Biotechnol. 14, 1246-1251) that a tightly packed structure exists in the protein. We also generated internal deletions within the loop regions of GFP according to its crystal structure and found that all such deletions eliminated GFP fluorescence.

Polyamine-mediated degradation of vertebrate ornithine decarboxylase (ODC) is associated with the production of antizyme, a reversible tightly binding protein inhibitor of ODC activity. The interaction of antizyme with a binding element near the N terminus of ODC is essential but not sufficient for regulation of the enzyme by polyamines (X. Li and P. Coffino, Mol. Cell. Biol. 12:3556-2562, 1992). We now show that a second element present at the C terminus is required for the degradation process. Antizyme caused a conformational change in ODC, which made the C terminus of ODC more accessible. Blocking the C terminus with antibody prevented degradation. Tethering the C terminus by creating a circularly permuted, enzymatically active form of ODC prevented antizyme-mediated degradation. These data elucidate a form of feedback regulation whereby excess polyamines induce destruction of ODC, the enzyme that initiates their biosynthesis.

Transcription factors (TFs) are critical regulators of cell growth and differentiation, whose dysfunction is associated with many human diseases, including cancer. To facilitate the discovery of functionally altered TFs among the approximately 2,000 human TFs, we (Panomics, Inc., Fremont, CA) developed a Protein/DNA array technology that can be used to profile the activities of multiple TFs simultaneously. In this study, we applied this technology to examine the TF activities in three different breast cancer cell lines: MCF7 (estrogen receptor [ER] +, tamoxifen-sensitive), T47D (ER+, tamoxifen-resistant), and HCC1806 (ER-, tamoxifen-resistant). We compared the differences in TF activity in these cells lines following treatment with estradiol or tamoxifen. We found a number of TF activities unique to each of these cell lines. In addition to verifying previous findings, the novel findings of this study provide a more comprehensive view of the differences in the response of these cancer lines to estrogen and tamoxifen.

Transcription factors are a group of proteins that modulate the expression of genes involved in many biological processes, such as cell growth and differentiation. Alterations in transcription factor function are associated with many human diseases, and therefore these proteins are attractive potential drug targets. A key issue in the development of such therapeutics is the generation of effective tools that can be used for high throughput discovery of the critical transcription factors involved in human diseases, and the measurement of their activities in a variety of disease or compound-treated samples. Here, a number of innovative arrays and 96-well format assays for profiling and measuring the activities of transcription factors will be discussed.

The glucocorticoid receptor (GR) regulates gene expression by binding specific sequence elements within the promoters of target genes or by cross-talk with other transcription factors (TFs). For some TFs, interaction with the GR results in alteration of DNA binding and transcriptional regulation. We used a protein-DNA array, a system that facilitates simultaneous profiling of the activities of multiple transcription factors, to systematically examine the potential cross-talk of GRalpha with 149 TFs. Using this array, we identified several TFs, including IRF, E47, and COUP-TF, whose DNA binding activities were modulated by GRalpha. We then confirmed these results with in vitro electrophoretic mobility shift assays and in vivo reporter assays. In this study, IRF and E47 were identified as participants in GRalpha cross-talk for the first time. This new finding expands our understanding of the functional role of GRalpha in the context of gene expression regulation.

First identified as part of the Rous sarcoma oncogene product Src, SH3 (Src Homology 3) domains play an important role in intercellular communication and intracellular signal transduction. A high-throughput assay for ligand binding to SH3 domains--SH3 domain proteins immobilized on a membrane--allows rapid visualization of numerous SH3 domain protein-protein interactions with no expensive equipment or radioactivity required. Once the array is constructed or obtained commercially, the procedure is straightforward: The protein of interest is cloned into a fusion-tagged expression vector and expressed in bacteria, the prepared bacterial extract is incubated with the array membrane, and the signal is measured using a chemiluminescence detection system.

Activation of the transcription factor NFkappaB requires rapid degradation of its inhibitor, IkappaBalpha. To facilitate the study of IkappaBalpha degradation, we fused IkappaBalpha protein to enhanced green fluorescent protein to construct IkappaBalpha-enhanced green fluorescent protein (IG). We demonstrated by both flow cytometry and Western blot analysis that the half-life of IG in the presence of human tumor necrosis factor (TNF) alpha is approximately 5 min, which is similar to the half-life of native IkappaBalpha. The degradation coincided with NFkappaB translocation from the cytoplasm to the nucleus and NFkappaB-mediated induction of transcription. Phorbol 12-myristate 13-acetate (PMA), but not forskolin, also induces degradation of IG fusion protein. The half-life of IG in the presence of PMA is approximately 15 min, longer than when induced with TNFalpha. Co-treatment with TNFalpha and PMA did not result in a synergistic effect on IG degradation, although they stimulate different kinases in two different signaling pathways. Degradation of IG was inhibited by mutations at serine residues 32 and 36, which are the target sites of the phosphorylation modification that initiates degradation of IkappaBalpha. We also demonstrated that basal degradation of IG in the presence of cycloheximide is inhibited by such mutations, suggesting that basal degradation of IkappaBalpha also requires phosphorylation as the signal for degradation. Finally, we showed that the rate of TNFalpha-induced degradation of IG remains almost constant throughout the cell cycle, except at the mitotic phase, in which IG degrades more slowly.

Publisher Summary The green fluorescent protein (GFP) from the jellyfish Aequorea victoria is a useful reporter, widely applied to the study of gene expression and protein localization in vivo. This wide application is because of the autofluorescent activity of the protein, which does not require any substrate or cofactor. Variants with mutations affecting the chromophore structure and the expression of the protein in human cells have been engineered; these mutations have enhanced the expression level and modified the fluorescence properties of GFP in mammalian cells. Enhanced GFP (EGFP) is such a mutant with a 35-fold increase in fluorescence intensity. Its enhanced fluorescence intensity makes EGFP a popular reporter in many applications. By fusion with other proteins, EGFP is widely used to monitor the expression and distribution of the target proteins in live cells. By linking it with a promoter/enhancer element, expression of EGFP can be used to measure the promoter activity as well as to determine its regulation. The introduction of EGFP into transgenic animals permits the study of gene expression and regulation of stage-dependent and spatially distributed genes. The limitation of EGFP as a reporter in certain cases is because of its stability. Crystallographic structures of GFP and its S65T mutant reveal a compacted β-barrel structure. This structure presumably contributes to its unusual resistance to proteolysis and denaturation. The stability of EGFP means a slower turnover in vivo, which limits its applications in certain cases, such as being a reporter in transient transcription studies.

Publisher Summary Screening of stable mammalian cell clones that express a target gene is often a rate-limiting step in establishing such cell lines for functional studies of gene expression. Generally speaking, the target gene needs to be cotransfected with an antibiotic resistance gene that is used for selecting the transfected cells. Among the selected clones, only a few express the target gene owing to the low efficiency of plasmid vector-mediated transfection. Therefore, a large number of drug-resistant colonies are needed for screening in order to obtain such a positive clone. Reverse transcription polymerase chain reaction (RT-PCR), Western blot analysis, and functional assays are often used to identify positive clones. Most of these screening procedures are tedious and time consuming. This chapter reports a simplified screening procedure to identify clones that express the target gene by co-expression with enhanced green fluorescent protein (EGFP), using a bidirectional vector. The target gene and EGFP are simultaneously expressed by the bidirectional vector. The target gene expression can be easily identified by screening for EGFP expression. EGFP is a mutant of wild-type GFP with a 35-fold increase in fluorescence, which is easily detected with a fluorescence microscope.

Regulated degradation of ornithine decarboxylase (ODC) is mediated by its association with the inducible protein antizyme. The N terminus of antizyme (NAZ), although unneeded for the interaction with ODC, must be present to induce degradation. We report here that covalently grafting NAZ to ODC confers lability that normally results from the non-covalent association of native antizyme and ODC. To determine whether NAZ could act similarly as a modular functional domain when grafted to other proteins, we fused it to a region of cyclin B (amino acids 13-90) capable of undergoing degradation or to cyclin B (amino acids 13-59), which is not subject to degradation. The association with NAZ made both NAZ-cyclin B13-90 and NAZ-cyclin B13-59 unstable. Furthermore, NAZ and cyclin B 13-59 were together able to induce in vitro degradation of Trypanosoma brucei ODC, a stable protein. The ODC-antizyme complex bound to the 26 S protease but not the 20 S proteasome, consistent with the observation that ODC degradation is mediated by the 26 S protease. The association was shown to be independent of NAZ, suggesting that NAZ does not act as a recognition signal.

Degradation provides one means for controlling the cellular level of the p53 tumor suppressor. Here we have determined a structural element of p53 required for degradation. To create a substrate amenable to in vitro analysis of proteolysis, we appended to p53 the N terminus of antizyme, a protein that binds to and induces degradation of mammalian ornithine decarboxylase (ODC). We found using deletion analysis that an element within amino acids 100-150 is required for degradation of the fusion protein. A monoclonal antibody (PAb246) that binds close to this region prevents the degradation induced by human papillomavirus 16 E6 protein. Furthermore, we found that amino acids 100-150 of p53 can function as an independent domain to induce Trypanosoma brucei ODC, a stable protein, to be degraded in vivo or, by cooperating with an antizyme binding domain of ODC, to confer polyamine-dependent regulation.

Ornithine decarboxylase (ODC) of African trypanosomes is an important target for anti-trypanosomal chemotherapy because of its remarkable stability in vivo. The in vivo activity and stability of mammalian ODC are regulated by polyamines. Polyamines induce antizyme, which inactivates ODC by tight association and promotes degradation of ODC by the mammalian 26 S proteasome. Here we found, in contrast to mammalian cells, that polyamines caused no reduction of ODC activity in Trypanosoma brucei. Mouse ODC expressed in T. brucei was also unaffected by exogenous polyamines, suggesting that a mammalian antizyme equivalent may be absent in T. brucei. The rat antizyme expressed in T. brucei was found capable of inhibiting mouse ODC activity by the formation of rat antizyme-mouse ODC complex. However, complex formation did not lead to degradation of mouse ODC in T. brucei. Further in vitro experiments suggested the presence of an inhibitory factor(s) in trypanosome, which interferes with the degradation of mouse ODC. We also demonstrated the presence of proteasomes in T. brucei. But the mobility of the trypanosomal proteasome on native gel is different from that of the mammalian proteasome. Thus, the absence of antizyme, the presence of inhibitory factor(s), and the differences between trypanosomal and mammalian proteasome may account for the stability of mouse ODC in T. brucei cells.

Selective degradation by proteasomes of ornithine decarboxylase, the initial enzyme in polyamine biosynthesis, is mediated by the polyamine-inducible protein antizyme. Antizyme binds to a region near the N terminus of ornithine decarboxylase (X. Li and P. Coffino, Mol. Cell. Biol. 12:3556-3562, 1992). This interaction induces a conformational change in ornithine decarboxylase that exposes its C terminus and inactivates the enzyme (X. Li and P. Coffino, Mol. Cell. Biol. 13:1487-1492, 1993). Here we show that the C-terminal half of antizyme alone can inactivate ornithine decarboxylase and alter its conformation, but it cannot direct degradation of the enzyme, either in vitro or in vivo. A portion of the N-terminal half of antizyme must be present to promote degradation.

Intracellular degradation of vertebrate ornithine decarboxylase (ODC) is accelerated by polyamines, the products of the pathway controlled by ODC. Antizyme, a reversible, tightly binding protein inhibitor of ODC activity, is believed to be involved in this process. Mouse and Trypanosoma brucei ODCs are structurally similar, but the trypanosome enzyme, unlike that of the mouse, is not regulated by intracellular polyamines when expressed in hamster cells (L. Ghoda, D. Sidney, M. Macrae, and P. Coffino, Mol. Cell. Biol. 12:2178-2185, 1992). We found that mouse ODC interacts with antizyme in vitro but trypanosome ODC does not. To localize the region necessary for binding, we made a series of enzymatically active chimeric mouse-trypanosome ODCs and tested them for antizyme interaction. Replacing residues 117 to 140 within the 461-amino-acid mouse ODC sequence with the equivalent region of trypanosome ODC disrupted both antizyme binding and in vivo regulation. Formation of an antizyme-ODC complex is therefore required for regulated degradation.

We have developed a screening technology for the identification of short-lived proteins. A green fluorescent protein (GFP)-fusion cDNA library was generated for monitoring degradation kinetics. Cells expressing a subset of the GFP-cDNA expression library were screened to recover those in which the fluorescence signal diminished rapidly when protein synthesis was inhibited. Thirty clones that met the screening criteria were characterized individually. Twenty-three (73%) proved to have a half-life of 4 hours or less.

A series of 5,8-dideaza analogues of folic acid, isofolic acid, aminopterin, and isoaminopterin were evaluated for inhibition of thymidylate synthase, TS, from mouse L1210 leukemia cells with 10-propargyl-5,8-dideazafolic acid, CB3717, 4a, as the reference inhibitor. These compounds were also tested as inhibitors of human dihydrofolate reductase, DHFR, obtained from WIL2 cells. None of the analogues studied were as potent as 4a toward TS; however, 9-methyl-5,8-dideazaisoaminopterin, 6d, was only 2.5-fold less effective. Compound 4a was prepared by direct alkylation of the di-tert-butyl ester of 5,8-dideazafolic acid followed by hydrolysis of the resulting diethyl ester, which resulted from concomitant transesterification. It was found to be identical with a sample of 4a prepared by earlier methodology by using a variety of spectroscopic techniques. Its isomer, 9-propargyl-5,8-dideazaisofolic acid, 4b, which was synthesized by an analogous approach, was found to be dramatically less inhibitory toward TS than 4a. Each of the 2,4-diamino derivatives, including those possessing an allyl or propargyl group at N9, was an excellent inhibitor of DHFR, having a level of potency similar to that of methotrexate, MTX. However, many of these 5,8-dideazaaminopterin analogues were far more inhibitory toward TS than MTX.