Characterization of recombinant chloroperoxidase, and F103A and C29H/C79H/C87H mutants

Mechanistically and structurally chloroperoxidase (CPO) occupies a unique niche among heme containing enzymes. Chloroperoxidase catalyzes a broad range of reactions, such as oxidation of organic substrates, dismutation of hydrogen peroxide, and mono-oxygenation of organic molecules. To expand the synthetic utility of CPO and to appreciate the important interactions that lead to CPO’s exceptional properties, a site-directed mutagenesis study was undertaken. ^ Recombinant CPO and CPO mutants were heterologously expressed in Aspergillus niger. The overall protein structure was almost the same as that of wild type CPO, as determined by UV-vis, NMR and CD spectroscopies. Phenylalanine103, which was proposed to regulate substrate access to the active site by restricting the size of substrates and to control CPO’s enantioselectivity, was mutated to Ala. The ligand binding affinity and most importantly the catalytic activity of F103A was dramatically different from wild type CPO. The mutation essentially eliminated the chlorination and dismutation activities but enhanced, 4-10 fold, the epoxidation, peroxidation, and N-demethylation activities. As expected, the F103A mutant displayed dramatically improved epoxidation activity for larger, more branched styrene derivatives. Furthermore, F103A showed a distinctive enantioselectivity profile: losing enantioselectivity to styrene and cis-β-methylstyrene; having a different configuration preference on α-methylstyrene; showing higher enantioselectivites and conversion rates on larger, more branched substrates. Our results show that F103 acts as a switch box that controls the catalytic activity, substrate specificity, and product enantioselectivity of CPO. Given that no other mutant of CPO has displayed distinct properties, the results with F103A are dramatic. ^ The diverse catalytic activity of CPO has long been attributed to the presence of the proximal thiolate ligand. Surprisingly, a recent report on a C29H mutant suggested otherwise. A new CPO triple mutant C29H/C79H/C87H was prepared, in which all the cysteines were replaced by histidine to eliminate the possibility of cysteine coordinating to the heme. No active form protein was isolated, although, successful transformation and transcription was confirmed. The result suggests that Cys79 and Cys87 are critical to maintaining the structural scaffold of CPO. ^ In vitro biodegradation of nanotubes by CPO were examined by scanning electron microscope method, but little oxidation was observed. ^

Maternal HIV-1 envelope-specific antibody responses and reduced risk of perinatal transmission.

Despite the wide availability of antiretroviral drugs, more than 250,000 infants are vertically infected with HIV-1 annually, emphasizing the need for additional interventions to eliminate pediatric HIV-1 infections. Here, we aimed to define humoral immune correlates of risk of mother-to-child transmission (MTCT) of HIV-1, including responses associated with protection in the RV144 vaccine trial. Eighty-three untreated, HIV-1-transmitting mothers and 165 propensity score-matched nontransmitting mothers were selected from the Women and Infants Transmission Study (WITS) of US nonbreastfeeding, HIV-1-infected mothers. In a multivariable logistic regression model, the magnitude of the maternal IgG responses specific for the third variable loop (V3) of the HIV-1 envelope was predictive of a reduced risk of MTCT. Neutralizing Ab responses against easy-to-neutralize (tier 1) HIV-1 strains also predicted a reduced risk of peripartum transmission in secondary analyses. Moreover, recombinant maternal V3-specific IgG mAbs mediated neutralization of autologous HIV-1 isolates. Thus, common V3-specific Ab responses in maternal plasma predicted a reduced risk of MTCT and mediated autologous virus neutralization, suggesting that boosting these maternal Ab responses may further reduce HIV-1 MTCT.

Antineoplastic Cytotoxicity and Immune Adjuvancy of a Recombinant Oncolytic Poliovirus

Our group has pioneered the development of a live-attenuated poliovirus, called PVSRIPO, for the purpose of targeting cancer. Despite clinical progress, the cancer selective cytotoxicity and immunotherapeutic potential of PVSRIPO has not yet been mechanistically dissected. Defining such mechanisms may inform its clinical application.

Herein I describe the discovery of a mechanism by which the MAP-Kinase Interacting Kinases (MNKs) regulate PVSRIPO cytotoxicity in cancer. In doing so, I delineate a novel, intricate network connecting the MNK and mTOR signaling pathway that regulates activity of a splicing kinase called the Ser-Arg Rich Protein Kinase (SRPK), and define SRPK as an impediment to IRES mediated translation. Moreover, I demonstrate that MNK regulates mTORC1 associations that determine its substrate proximity and thus, activity. In a collaborative effort, we found that PVSRIPO oncolysis produces antigen specific, cytolytic anti-tumor immunity in an in vitro human system and that much of the observed adjuvancy is due to the direct infection of dendritic cells (DCs) by the virus itself; implicating PVSRIPO as a potent adjuvant. In summary, oncogenic signaling in part through MNK leads to cancer specific cytotoxicity by PVSRIPO that engages an inflammatory environment conducive to DC activation and antigen specific T cell antigen immunity.

Characterization of hepcidin response to holotransferrin in novel recombinant TfR1 HepG2 cells

Hepcidin is the key regulator of systemic iron homeostasis. The iron-sensing mechanisms and the role of intracellular iron in modulating hepatic hepcidin secretion are unclear. Therefore, we created a novel cell line, recombinant-TfR1 HepG2,expressing iron-response-element-independent TFRC mRNA to promote cellular iron overload and examined the effect of excess holotransferrin (5 g/L) on cell-surface TfR1, iron content, hepcidin secretion and mRNA expressions of TFRC, HAMP, SLC40A1,HFE and TFR2. Results showed that the recombinant cells exceeded levels of cell surface TfR1 in wild-type cells under basal (2.8-fold; p<0.03) and holotransferrin supplemented conditions for 24 h and 48 h (4.4- and 7.5-fold, respectively; p<0.01). Also, these cells showed higher intracellular iron content than wild-type cells under basal (3-fold; p<0.03) and holotransferrin-supplemented conditions (6.6-fold at 4 h; p<0.01). However, hepcidin secretion was not higher than wild-type cells. Moreover, holotransferrin treatment to recombinant cells did not elevate HAMP responses compared to untreated or wild-type cells. In conclusion, increased intracellular iron content in recombinant cells did not increase hepcidin responses compared to wild-type cells, resembling hemochromatosis. Furthermore, TFR2 expression altered within 4 h of treatment, while HFE expression altered later at 24 h and 48 h, suggesting that TFR2 may function prior to HFE in HAMP regulation.

Data for the co-expression and purification of human recombinant CaMKK2 in complex with calmodulin in Escherichia coli

Calcium/calmodulin-dependent kinase kinase 2 (CaMKK2) has been implicated in a range of conditions and pathologies from prostate to hepatic cancer. Here, we describe the expression in Escherichia coli and the purification protocol for the following constructs: full-length CaMKK2 in complex with CaM, CaMKK2 'apo', CaMKK2 (165-501) in complex with CaM, and the CaMKK2 F267G mutant. The protocols described have been optimized for maximum yield and purity with minimal purification steps required and the proteins subsequently used to develop a fluorescence-based assay for drug binding to the kinase, "Using the fluorescent properties of STO-609 as a tool to assist structure-function analyses of recombinant CaMKK2"

Stable expression of a human-like sialylated recombinant thyrotropin in a chinese hamster ovary cell line expressing 'alfa'2,6-sialyltransferase

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Avaliação do padrão de resposta de imunoglobulinas em diferentes linhagens de camundongos frente à infecção por T.cruzi

Dissertação (Mestrado)

Effets de l’environnement lumineux et de l’âge foliaire sur la croissance, la capacité photosynthétique et la production protéique chez "Nicotiana benthamiana"

Cette étude visait à caractériser la croissance, la capacité photosynthétique, la concentration en azote et protéines totales solubles, la production de protéines recombinantes (HA) ainsi que la quantité de lumière interceptée à différents stades de développement de plants de Nicotiana benthamiana afin d’optimiser la production de vaccins. L’évolution des réponses physiologiques étudiées fut similaire chez toutes les feuilles primaires, suggérant que le processus de sénescence s’initie et progresse de façon semblable indépendamment de leur ordre d’initiation. Toutefois, la superposition des patrons temporels de sénescence et de croissance foliaire a mené à un rendement HA maximal se situant invariablement dans la partie médiane du plant lorsqu’exprimé sur une base foliaire. À l’échelle du plant entier, nos résultats suggèrent qu’il est possible d’augmenter la production de vaccins en récoltant les plants à un stade de développement plus tardif, ou en augmentant la densité de culture et en récoltant ces plants plus tôt.

Protection of white leg shrimp (Litopenaeus vannamei) against white spot disease virus using oral recombinant bioencapsulated VP28

Aquaculture, is perceived as having the greatest potential to meet the growing demand for aquatic food. Crustaceans form one of the main value added components in aquaculture and among them, shrimp aquaculture is the predominant one. Industrial shrimp fanning, in combination with poor management in shrimp aquaculture, has quickly led to severe pollution in shrimp ponds, thereby creating a suitable environment for development of bacterial and virus diseases. White spot disease is one of the most deadly diseases that are caused heavy loss in all Penaeid shrimps family. In Iran during 2002 to 2004 in the Kuzestan province and in 2005 in Bushehr province, the most ponds and farms infected with white spot and the entire industry was facing threat of closure. Owing to the impact of WSSV infection to shrimp aquaculture, there is an urgent need to develop suitable strategies to protect cultured shrimps and make aquaculture more sustainable. Therefore, this study aimed to examine the possibility of protecting shrimp against white spot syndrome virus using bioencapsulated Anemia with E. coil containing the recombinant protein VP28, designed. Virus genome was extracted from naturally infected Litopenaeus vannamei in the Choebdch farms and VP28 gene by designed primers was amplified, extracted, purified and cloned in E. coli TGI. Protein expression evaluated and inactivated bacteria containing recombinant protein encapsulated in Artemia nauplii. White shrimp post larvae stage 5 were fed for 5 days with recombinant nauplii and twice on days 7 and 25 after feeding with Artemia nauplii were challenged with white spot virus. The results of the first experiment revealed that cumulative mortality percent in the group receiving the bacteria containing recombinant plasmid (pMal + VP28) was %14.44±1.11 and the relative percent survival %80.30±1.51. In this group the mortality rates in the various repetitions varied from the 13.33% to 16.66% and relative percent survival of 77.27% to 81.81%. in the Non-recombinant plasmid group (pMal) Mean percent mortality was% 33.33±3.84 and the Relative Percent Survival %54.54±5.24 and in the group that received bacteria contained no recombinant plasmid the Mean cumulative mortality percent was%48.88 ± 5.87 and Relative Percent Survival%33.33± 8.01.

Recombinant human endostatin reduces hypertrophic scar formation in rabbit ear model through down- regulation of VEGF and TIMP-1.

Background: Recombinant human endostatin (Endostar) has been widely used to suppress angiogenesis in carcinoma patients. Hypertrophic scar (HS) tissue, much like a carcinoma, is often associated with angiogenesis. However, there have been few studies conducted on the effects of Endostar on HS or its mechanism. Objective: This paper investigated the effects Endostar on the HS of rabbit ears and studied the effects of Endostar on VEGF and TIMP-1 expression. Methods: Sixteen New Zealand white rabbits were used to establish HS models. Then, rabbit ears containing HS were randomly assigned to either the Endostar group or the control group. The changes of appearance and histology were evaluated using the naked eye, hematoxylin eosin staining, and a scar elevation index. The VEGF and TIMP-1 expressions were detected by immunohistochemical staining, RT-PCR, and western blot. Results: The thickness of the connective tissue in the Endostar group were thinner, the numbers of micro vessels and fibroblasts were fewer, and the collagen fibers were smoother. Moreover, the mRNA and protein expressions of VEGF and TIMP-1 in the Endostar group were significantly lower than those in the control group. Conclusion: The results suggested that Endostar reduced the formation of HS by down-regulation of VEGF and TIMP-1 expressions.