Single step recombinant human growth hormone (rhGH) purification from milk by peptide affinity chromatography

Recombinant human growth hormone (rhGH) is used for the treatment of several pathologies, most of them related to growth. Although different expression systems can be used for its production, the milk from transgenic cows is one of the most interesting due to the high rhGH level achieved (5 g/L). We have designed and synthesized short peptides (9 or 10 amino acid long) using Fmoc chemistry and studied their ability to purify rhGH from milk once immobilized on an agarose support. Using spiked milk with the hormone as a sample, rhGH was purified with 88% yield and 92% purity in a single step with a fold purification of 4.5. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:999–1005, 2018     

Joint effects of germ-line <Emphasis Type="Italic">TP53</Emphasis> mutation, <Emphasis Type="Italic">MDM2</Emphasis> SNP309, and gender on cancer risk in family studies of Li–Fraumeni syndrome

Li–Fraumeni syndrome (LFS) is a rare familial cancer syndrome characterized by early cancer onset, diverse tumor types, and multiple primary tumors. Germ-line TP53 mutations have been identified in most LFS families. A high-frequency single-nucleotide polymorphism, SNP309 (rs2279744), in MDM2 was recently confirmed to be a modifier of cancer risk in several case-series studies: substantially earlier cancer onset was observed in SNP309 G-allele carriers than in wild-type individuals by 7–16 years. However, cancer risk analyses that jointly account for measured hereditary TP53 mutations and MDM2 SNP309 have not been systematically investigated in familial cases. Here, we determined the combined effects of measured TP53 mutations, MDM2 SNP309, and gender and their interactions simultaneously in LFS families. We used the method that is designed for extended pedigrees and structured for age-specific risk models based on Cox proportional hazards regression. We analyzed the cancer incidence in 19 extended pedigrees with germ-line TP53 mutations ascertained through the clinical LFS phenotype. The dataset consisted of 463 individuals with 129 TP53 mutation carriers. Our analyses showed that the TP53 germ-line mutation and its interaction with gender were strongly associated with familial cancer incidence and that the association between MDM2 SNP309 and increased cancer risk was modest. In contrast with several case-series studies, the interaction between MDM2 SNP309 and TP53 mutation was not statistically significant in our LFS family cohort. Our results showed that SNP309 G-alleles were associated with accelerated tumor formation in both carriers and non-carriers of germ-line TP53 mutations.     

Mdm2 and Mdm4 loss regulates distinct p53 activities

Mutational inactivation of p53 is a hallmark of most human tumors. Loss of p53 function also occurs by overexpression of negative regulators such as MDM2 and MDM4. Deletion of Mdm2 or Mdm4 in mice results in p53-dependent embryo lethality due to constitutive p53 activity. However, Mdm2(-/-) and Mdm4(-/-) embryos display divergent phenotypes, suggesting that Mdm2 and Mdm4 exert distinct control over p53. To explore the interaction between Mdm2 and Mdm4 in p53 regulation, we first generated mice and cells that are triple null for p53, Mdm2, and Mdm4. These mice had identical survival curves and tumor spectrum as p53(-/-) mice, substantiating the principal role of Mdm2 and Mdm4 as negative p53 regulators. We next generated mouse embryo fibroblasts null for p53 with deletions of Mdm2, Mdm4, or both; introduced a retrovirus expressing a temperature-sensitive p53 mutant, p53A135V; and examined p53 stability and activity. In this system, p53 activated distinct target genes, leading to apoptosis in cells lacking Mdm2 and a cell cycle arrest in cells lacking Mdm4. Cells lacking both Mdm2 and Mdm4 had a stable p53 that initiated apoptosis similar to Mdm2-null cells. Additionally, stabilization of p53 in cells lacking Mdm4 with the Mdm2 antagonist nutlin-3 was sufficient to induce a cell death response. These data further differentiate the roles of Mdm2 and Mdm4 in the regulation of p53 activities.     

Mutant p53 disrupts role of ShcA protein in balancing Smad protein-dependent and -independent signaling activity of transforming growth factor-β (TGF-β)

Biomarkers are lacking for identifying the switch of transforming growth factor-β (TGF-β) from tumor-suppressing to tumor-promoting. Mutated p53 (mp53) has been suggested to switch TGF-β to a tumor promoter. However, we found that mp53 does not always promote the oncogenic role of TGF-β. Here, we show that endogenous mp53 knockdown enhanced cell migration and phosphorylation of ERK in DU145 prostate cancer cells. Furthermore, ectopic expression of mp53 in p53-null PC-3 prostate cancer cells enhanced Smad-dependent signaling but inhibited TGF-β-induced cell migration by down-regulating activated ERK. Reactivation of ERK by the expression of its activator, MEK-1, restored TGF-β-induced cell migration. Because TGF-β is known to activate the MAPK/ERK pathway through direct phosphorylation of the adaptor protein ShcA and MAPK/ERK signaling is pivotal to tumor progression, we investigated whether ShcA contributed to mp53-induced ERK inhibition and the conversion of the role of TGF-β during carcinogenesis. We found that mp53 expression led to a decrease of phosphorylated p52ShcA/ERK levels and an increase of phosphorylated Smad levels in a panel of mp53-expressing cancer cell lines and in mammary glands and tumors from mp53 knock-in mice. By manipulating ShcA levels to regulate ERK and Smad signaling in human untransformed and cancer cell lines, we showed that the role of TGF-β in regulating anchorage-dependent and -independent growth and migration can be shifted between growth suppression and migration promotion. Thus, our results for the first time suggest that mp53 disrupts the role of ShcA in balancing the Smad-dependent and -independent signaling activity of TGF-β and that ShcA/ERK signaling is a major pathway regulating the tumor-promoting activity of TGF-β.     

Cadmium removal capacities of filamentous soil fungi isolated from industrially polluted sediments,in La Plata (Argentina)

Aspergillus terreus, Cladosporium cladosporioides, Fusarium oxysporum, Gliocladium roseum, Penicillium spp., Talaromyces helicus and Trichoderma koningii were isolated from heavily polluted streams near an industrial area in La Plata, Argentina. The fungi were obtained from sediments with 0.25–0.50 mg Cd/l and they were isolated in cadmium-basal medium. They were then cultivated to evaluate their Cd detoxification abilities. The biomass developed in static assays represented 5–53% of the yield of stirred cultures, for the different fungal species, although the cadmium absorption were similar in both cases. These soil fungi represented 50% of the total isolates and their mycelial growth was conspicuous in these polluted sediments. Although bacteria have been mentioned as active microorganisms against heavy metals, the mycelial fungi were able to develop a significantly higher mass to sequestrate more metals. Thus, they could be used in remediation biotechnology to improve the Cd detoxification of chronically contaminated habitats.     

Molecular and kinetic characterization of mixed cultures degrading natural and synthetic estrogens

The biodegradation of estradiol (E2), estrone (E1), and ethinylestradiol (EE2) was investigated using mixed bacterial cultures enriched from activated sludge. Enrichments were carried out on E2 or EE2 in batch conditions with acetonitrile as additional carbon source. Degradation experiments were performed both using hormones as sole carbon source or with an additional source. The hormones were completely degraded by these cultures. Estradiol was rapidly converted to E1 within 24 h. Thereafter, E1 degradation began, displaying a lag phase ranging from 3 to 4 days. Estrone depletion took from 48 h to more than 6 days, depending on the culture conditions. For EE2 degradation, when it was the sole carbon source, the lag phase and the time required for its complete removal (7 and 15 days, respectively) were shorter that in cultures with a supplementary carbon source. The specific degradation rates observed for E2 both with and without an additional carbon source were similar. By contrast, the specific degradation rates for E1 and EE2 were, respectively, seven and 20 times faster when these hormones were supplied as the sole carbon source. The bacterial community structure of each culture was characterized by molecular and cultural methods. The mixed cultures were made up of species belonging to Alcaligenes faecalis, Pusillimonas sp., Denitrobacter sp., and Brevundimonas diminuta or related to uncultured Bacteroidetes. The isolated strain B. diminuta achieved the conversion of E2 to E1.     

Multimeric system of 99mTc-labeled gold nanoparticles conjugated to c[RGDfK(C)] for molecular imaging of tumor α(v)β(3) expression

Integrin α(V)β(3) plays a critical role in tumor angiogenesis and metastasis. Suitably radiolabeled cyclic RGD peptides can be used for noninvasive imaging of α(V)β(3) expression. The aim of this research was to prepare a multimeric system of technetium-99m-labeled gold nanoparticles conjugated to c[RGDfK(C)] and to evaluate its biological behavior as a potential radiopharmaceutical for molecular imaging of tumor angiogenesis. Hydrazinonicotinamide-GGC (HYNIC-GGC) and c[RGDfK(C)] peptides were synthesized and conjugated to gold nanoparticles (AuNP, 20 nm) by means of spontaneous reaction of the thiol groups of cysteine. The nanoconjugate was characterized by TEM, FT-IR, UV-vis, XPS, and Raman spectroscopy. To obtain (99m)Tc-HYNIC-GGC-AuNP-c[RGDfK(C)] ((99m)Tc-AuNP-RGD), the (99m)Tc-HYNIC-GGC radiopeptide was first prepared and added to 1.5 mL of AuNP solution (1 nM) followed by c[RGDfK(C)] (10 μL, 50 μM) at 18 °C with stirring for 15 min. Radiochemical purity (RP) was determined by size-exclusion HPLC and ITLC-SG analyses. In vitro binding studies were carried out in α(V)β(3) receptor-positive C6 glioma cancer cells. Biodistribution studies were accomplished in athymic mice with C6-induced tumors with blocked and nonblocked receptors, and images were obtained using a micro-SPECT/CT. TEM and spectroscopy techniques demonstrated that AuNPs were functionalized with peptides. RP was 96 ± 2% without postlabeling purification. (99m)Tc-AuNP-RGD showed specific recognition for α(V)β(3) integrins expressed in C6 cells, and 3 h after i.p. administration in mice, the tumor uptake was 8.18 ± 0.57% ID/g. Micro-SPECT/CT images showed evident tumor uptake. (99m)Tc-AuNP-RGD demonstrates properties suitable for use as a target-specific agent for molecular imaging of tumor α(V)β(3) expression.     

CPT1c is localized in endoplasmic reticulum of neurons and has carnitine palmitoyltransferase activity

CPT1c is a carnitine palmitoyltransferase 1 (CPT1) isoform that is expressed only in the brain. The enzyme has recently been localized in neuron mitochondria. Although it has high sequence identity with the other two CPT1 isoenzymes (a and b), no CPT activity has been detected to date. Our results indicate that CPT1c is expressed in neurons but not in astrocytes of mouse brain sections. Overexpression of CPT1c fused to the green fluorescent protein in cultured cells demonstrates that CPT1c is localized in the endoplasmic reticulum rather than mitochondria and that the N-terminal region of CPT1c is responsible for endoplasmic reticulum protein localization. Western blot experiments with cell fractions from adult mouse brain corroborate these results. In addition, overexpression studies demonstrate that CPT1c does not participate in mitochondrial fatty acid oxidation, as would be expected from its subcellular localization. To identify the substrate of CPT1c enzyme, rat cDNA was overexpressed in neuronal PC-12 cells, and the levels of acylcarnitines were measured by high-performance liquid chromatography-mass spectrometry. Palmitoylcarnitine was the only acylcarnitine to increase in transfected cells, which indicates that palmitoyl-CoA is the enzyme substrate and that CPT1c has CPT1 activity. Microsomal fractions of PC-12 and HEK293T cells overexpressing CPT1c protein showed a significant increase in CPT1 activity of 0.57 and 0.13 nmol.mg(-1).min(-1), respectively, which is approximately 50% higher than endogenous CPT1 activity. Kinetic studies demonstrate that CPT1c has similar affinity to CPT1a for both substrates but 20-300 times lower catalytic efficiency.