High-level expression of a recombinant active microbial transglutaminase in Escherichia coli

Background: Bacterial transglutaminases are increasingly required as industrial reagents for in vitro modification of proteins in different fields such as in food processing as well as for enzymatic site-specific covalent conjugation of therapeutic proteins to polyethylene glycol to get derivatives with improved clinical performances. In this work we studied the production in Escherichia coli of a recombinant transglutaminase from Streptomyces mobaraensis (microbial transglutaminase or MTGase) as enzymatically active chimeric forms using different expression systems under the control of both lac promoter or thermoinducible phage lambda promoter. Results: Thermoinducible and constitutive expression vectors were constructed expressing Met-MTGase with chimeric LacZ 1-8 PNP 1–20 or LacZ 1–8 fusion protein under different promoters. After transformed in competent Escherichia coli K12 strains were fermented in batch and fed-bach mode in different mediums in order to select the best conditions of expression. The two most performing fusion protein systems namely short thermoinducible LacZ 1–8 Met-MTGase from NP668/1 and long constitutive LacZ 1-8 PNP 1–20 Met-MTGase from NP650/1 has been chosen to compare both efficiency of expression and biochemical qualities of the product. Proteins were extracted, purified to homogeneity and verified as a single peak obtained in RP-HPLC. The LacZ 1-8 PNP 1–20 Met-MTGase fusion protein purified from NP650/1 exhibited an activity of 15 U/mg compared to 24 U/mg for the shorter fusion protein purified from NP668/1 cell strain. Conclusions: Combining the experimental data on expression levels and specific activities of purified MTGase fusion proteins, the chimeric LacZ 1-8 Met-MTGase, which displays an enzymatic activity comparable to the wild-type enzyme, was selected as a candidate for producing microbial transglutaminase for industrial applications.Pubblicat

The beneficial effect of Zinc(II) on low-dose chemotherapeutic sensitivity involves p53 activation in wild-type p53-carrying colorectal cancer cells

BACKGROUND: Activation of wild-type p53 in response to genotoxic stress occurs through different mechanisms including protein conformation, posttranslational modifications, and nuclear localization, leading to DNA binding to sequence-specific promoters. Zinc ion plays a crucial role in stabilizing p53/DNA binding to induce canonical target genes. Mutant p53 proteins undergo protein misfolding that can be counteracted by zinc. However, whether zinc supplementation might have a beneficial antitumor effect in wild-type p53-carrying cells in combination with drugs, has not been addressed so far. METHODS: In this study we compared the effect of two antitumor treatments: on the one hand wild-type p53-carrying colon cancer cells were treated with low and high doses of chemotherapeutic agent Adriamycin and, on the other hand, Adriamycin was used in combination with ZnCl2. Biochemical and molecular analyses were applied to evaluate p53 activity and biological outcomes in this setting. Finally, the effect of the different combination treatments were applied to assess tumor growth in vivo in tumor xenografts. RESULTS: We found that low-dose Adriamycin did not induce p53 activation in wtp53-carrying colon cancer cells, unless in combination with ZnCl2. Mechanistically, ZnCl2 was a key determinant in inducing wtp53/DNA binding and transactivation of target genes in response to low-dose Adriamycin that used alone did not achieve such effects. Finally, in vivo studies, in a model of wtp53 colon cancer xenograft, show that low-dose Adriamycin did not induce tumor regression unless in combination with ZnCl2 that activated endogenous wtp53. CONCLUSIONS: These results provide evidence that ZnCl2 might be a valuable adjuvant in chemotherapeutic regimens of colorectal cancer harboring wild-type p53, able to both activate p53 and reduce the amount of drugs for antitumor purposes

Dermal Papilla Cells improve the wound healing process and generate hair bud-like structures in grafted skin substitutes using Hair Follicle Stem Cells

Tissue-engineered skin represents a useful strategy for the treatment of deep skin injuries and may contribute to the understanding of skin regeneration. The growth of hair follicles in vitro or after grafting remains a major challenge. The dermal-epidermal composites are skin substitutes comprised of dermal fibroblasts (DF) embedded in a matrix overlaid with keratinocytes. Hair Follicle Stem Cells (HFSC) contribute to hair follicle regeneration and wound repair. Dermal papilla cells (DPC) signaling orchestrates hair follicle morphogenesis and regeneration. The use of DPC as dermal component in a permanent composite skin with human HFSC was evaluated by studying tissue-engineered skin architecture, stem cell persistence and hair regeneration as well as the graft-take in nude mice. A porcine acellular dermal matrix (ADM) was seeded with HFSC alone and with human DPC or DF. Histological and immunohistochemical analyses of in vitro constructs were performed. The presence of DPC induced a more regular and multi-layered stratified epidermis with more basal p63-positive cells and invaginations. Graft-take and tissue remodeling in nude mice were favored in DPC-containing composite skin supported by the fact of graft-epidermis survival and early neovascularization. Interestingly, only in grafted constructs containing DPC, embryonic hair bud-like structures were observed from 14 days after grafting. These structures showed cells of human origin, presence of precursor epithelial cells and expression of a hair differentiation marker. These observations suggest an incipient hair follicle neogenesis inside the remodeling ADM. Taken together our results show DPC and HFSC as promising cellular components for a permanent skin substitute.Fil: Leiros, Gustavo Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencias y Tecnología "Dr. Cesar Milstein"; ArgentinaFil: Kusinsky, Ana Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencias y Tecnología "Dr. Cesar Milstein"; ArgentinaFil: Drago, Hugo. Hospital de Quemados de la Ciudad de Buenos Aires. Banco de Tejidos; ArgentinaFil: Bossi, Silvia. Hospital de Quemados de la Ciudad de Buenos Aires. Banco de Tejidos; ArgentinaFil: Sturla, Flavio. Hospital de Quemados de la Ciudad de Buenos Aires. Banco de Tejidos; ArgentinaFil: Castellanos, Maria Lia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencias y Tecnología "Dr. Cesar Milstein"; ArgentinaFil: Stella, Inés Yolanda. Universidad Maimónides. Centro de Estudios Biomédicos, Ambientales y Diagnóstico; ArgentinaFil: Balaña, Maria Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencias y Tecnología "Dr. Cesar Milstein"; Argentin

Biological heterogeneity of putative bladder cancer stem-like cell populations from human bladder transitional cell carcinoma samples.

Transitional cell carcinoma (TCC) is the most common type of bladder cancer. Emerging evidence has suggested that the capability of a tumor to grow and propagate is dependent on a small subset of cells, the cancer stem-like cells (CSCs) or tumor initiating cells. We report on the isolation and biological characterization of putative bladder CSC populations from primary TCCs. Isolated cells were induced to proliferate in stem cell culture conditions (serum-free medium containing mitogenic growth factors). The proliferating cells formed spheroids (urospheres) and their abilities for extensive proliferation and self-renewal were assayed. Their positivity for several stem cell markers (CD133, Oct-3/4, nestin, and cytokeratins) was also assessed by immunofluorescence tests and they could have the potential to differentiate in the presence of serum. In stem cell culture conditions they gradually showed loss of proliferation, adherence to the substrate, and morphological changes, which might reflect their progressive acquisition of differentiative capacity and loss of self-renewal ability. To evaluate if effective cell selection occurred after isolation, conventional cytogenetic studies on fresh chromosome spreads immediately after isolation and after culture were carried out. In addition, a molecular cytogenetic study by UroVysion assay was carried out on paraffin-embedded tissue sections and on fresh and after culture nuclei preparations. The data collected indicated important karyotype changes and a positive selection for hypo- or near-diploid cells, losing the complexity present in fresh tumors

Association of CYP1A1 A4889G and T6235C polymorphisms with the risk of sporadic breast cancer in Brazilian women

OBJECTIVES:We examined the influence of CYP1A1 A4889G and T6235C polymorphisms on the risk of sporadic breast cancer.METHODS:DNA from 742 sporadic breast cancer patients and 742 controls was analyzed using the polymerase chain reaction, followed by the restriction fragment length polymorphism technique.RESULTS:More patients had the CYP1A1 4889AG+GG genotype compared to controls (29.0% versus 23.2%, p=0.004). The G allele carriers had a 1.50-fold increased risk (95% CI: 1.14-1.97) of sporadic breast cancer compared to the other study participants. The frequency of the 4889AG+GG genotype among the Caucasian patients was higher than in the non-Caucasian patients (30.4% versus 20.2%, p=0.03) and controls (30.4% versus 23.2%, p=0.002). Caucasians and G allele carriers had a 1.61-fold increased risk (95% CI: 1.20-2.15) of sporadic breast cancer compared to other subjects. The CYP1A1 4889AG+GG genotype was more common among patients with a younger median age at first full-term pregnancy than among controls (33.8% versus 23.2%, p=0.001) and subjects whose first full-term pregnancies occurred at an older age (33.8% versus 26.1%, p=0.03). Women with the CYP1A1 4889AG+GG genotype and earlier first full-term pregnancies had a 1.87-fold (95% CI: 1.32-2.67) increased risk of sporadic breast cancer compared to the other study participants. Excess CYP1A1 4889AG+GG (39.8% versus27.1%, p=0.01) and 6235TC+CC (48.4% versus 35.9%, p=0.02) genotypes were also observed in patients with grade I and II tumors compared to patients with grade III tumors and controls (39.8% versus 23.2%, p=0.04; 48.4% versus 38.6%, p=0.04). The G and C allele carriers had a 2.44-fold (95% CI: 1.48-4.02) and 1.67-fold (95% CI: 1.03-2.69) increased risk, respectively, of developing grade I and II tumors compared to other subjects.CONCLUSIONS:The CYP1A1 A4889G and T6235C polymorphisms may alter the risk of sporadic breast cancer in Brazilian women

Association of CYP1A1 A4889G and T6235C polymorphisms with the risk of sporadic breast cancer in brazilian women

We examined the influence of CYP1A1 A4889G and T6235C polymorphisms on the risk of sporadic breast cancer. DNA from 742 sporadic breast cancer patients and 742 controls was analyzed using the polymerase chain reaction, followed by the restriction fragment length polymorphism technique. RESULTS: More patients had the CYP1A1 4889AG + GG genotype compared to controls (29.0% versus 23.2%, p=0.004). The G allele carriers had a 1.50-fold increased risk (95% CI: 1.14-1.97) of sporadic breast cancer compared to the other study participants. The frequency of the 4889AG + GG genotype among the Caucasian patients was higher than in the non-Caucasian patients (30.4% versus 20.2%, p=0.03) and controls (30.4% versus 23.2%, p=0.002). Caucasians and G allele carriers had a 1.61-fold increased risk (95% CI: 1.20-2.15) of sporadic breast cancer compared to other subjects. The CYP1A1 4889AG + GG genotype was more common among patients with a younger median age at first full-term pregnancy than among controls (33.8% versus 23.2%, p=0.001) and subjects whose first full-term pregnancies occurred at an older age (33.8% versus 26.1%, p=0.03). Women with the CYP1A1 4889AG + GG genotype and earlier first full-term pregnancies had a 1.87-fold (95% CI: 1.32-2.67) increased risk of sporadic breast cancer compared to the other study participants. Excess CYP1A1 4889AG + GG (39.8% versus 27.1%, p=0.01) and 6235TC + CC (48.4% versus 35.9%, p=0.02) genotypes were also observed in patients with grade I and II tumors compared to patients with grade III tumors and controls (39.8% versus 23.2%, p=0.04; 48.4% versus 38.6%, p=0.04). The G and C allele carriers had a 2.44-fold (95% CI: 1.48-4.02) and 1.67-fold (95% CI: 1.03-2.69) increased risk, respectively, of developing grade I and II tumors compared to other subjects. The CYP1A1 A4889G and T6235C polymorphisms may alter the risk of sporadic breast cancer in Brazilian women.7010680685FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPES

Cadaveric bone marrow mesenchymal stem cells: first experience treating a patient with large severe burns

Background: In January 2005, Rasulov et al. originally published ?First experience in the use of bone marrowmesenchymal stem cells (MSCs) for the treatment of a patient with deep skin burns. Here, we present the firstever treated patient with cadaveric bone marrow mesenchymal stem cells (CMSCs) in the history of Medicine.Methods: A young man, who severely burned 60 % of his total body surface with 30 % of full-thickness burns whileworking with a grass trimmer that exploded, was involved in the study. MSCs were obtained from the bone marrow ofa cadaver donor in a routine procurement procedure of CUCAIBA, the Province of Buenos Aires, Argentina, Ministry ofHealth, Transplantation Agency, cultured, expanded, and applied on the burned surfaces using a fibrin spray after earlyescharotomy.Results: So far, our preliminary experience and our early results have been very impressive showing an outstandingsafety data as well as some impressive good results in the use of CMSCs.Conclusions: Based on all this, we think that improvements in the use of stem cells for burns might be possible in thenear future and a lot of time as well as many lives could be saved by many other research teams all over the world.CMSCs will probably be a real scientific opportunity in Regenerative Medicine as well as in Transplantation.Fil: Mansilla, Eduardo. No especifíca;Fil: Marin, Gustavo Horacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; ArgentinaFil: Berges, Mirta. Hospital San Martin; ArgentinaFil: Scafatti, Silvia. Hospital San Martin; ArgentinaFil: Rivas, Jaime. Hospital San Martin; ArgentinaFil: Núñez, Andrea. Hospital San Martin; ArgentinaFil: Menvielle, Martin. Hospital San Martin; ArgentinaFil: Lamonega, Roberto. Hospital San Martin; ArgentinaFil: Gardiner, Cecilia. Hospital San Martin; ArgentinaFil: Drago, Hugo. Hospital Burns; ArgentinaFil: Sturla, Flavio. Hospital Burns; ArgentinaFil: Portas, Mercedes. Hospital Burns; ArgentinaFil: Bossi, Silvia. Hospital Burns; ArgentinaFil: Castuma, Maria Victoria. No especifíca;Fil: Peña Luengas, Sandra. Universidad de Puerto Rico; Puerto RicoFil: Roque, Gustavo. No especifíca;Fil: Martire, Karina. No especifíca;Fil: Taulamet, María José. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; ArgentinaFil: Orlandi, Gabriel. No especifíca;Fil: Tarditti, Adrian. No especifíca

Untailored vs. Gender- and Body-Mass-Index-Tailored Skeletal Muscle Mass Index (SMI) to Assess Sarcopenia in Advanced Head and Neck Squamous Cell Carcinoma (HNSCC)

: (1) background: sarcopenia lasting >1 year might be considered a chronic condition in many HNSCC patients. CT-scan-derived skeletal muscle mass Index (SMI) is an established surrogate of sarcopenia; yet, the cut-off reported in the literature (literature-based, lb-SMI < 43.2) is mainly based on the risk of chemoradiotherapy-induced toxicity, and the optimal value to discriminate OS is under-investigated. (2) methods: the effect on OS of the lb-SMI cutoff was compared with an untailored OS-oriented SMI cutoff obtained in a cohort of consecutive advanced HNSCC patients treated with primary chemoradiotherapy, bio-chemotherapy or chemo-immunotherapy (cohort-specific, cs-SMI cutoff). gender- and BMI-tailored (gt-SMI and bt-SMI) cut-offs were also evaluated. cutoff values were identified by using the maximally selected rank statistics for OS. (3) results: In 115 HNSCC patients, the cs-SMI cutoff was 31.50, which was lower compared to the lb-SMI reported cut-off. the optimal cut-off separately determined in females, males, overweight and non-overweight patients were 46.02, 34.37, 27.32 and 34.73, respectively. gt-SMI categorization had the highest effect on survival (p < 0.0001); its prognostic value was independent of the treatment setting or the primary location and was retained in a multivariate cox-regression analysis for OS including other HNSCC-specific prognostic factors (p = 0.0004). (4) conclusions: a tailored SMI assessment would improve clinical management of sarcopenia in chemoradiotherapy-, bio-chemotherapy- or chemo-immunotherapy-treated HNSCC patients. gender-based SMI could be used for prognostication in HNSCC patients