Purification of chondroitin precursor from Escherichia coli K4 fermentation broth using membrane processing

Recently the possibility of producing the capsular polysaccharide K4, a fructosylated chondroitin, in fed-batch experiments was assessed. In the present study, a novel downstream process to obtain chondroitin from Escherichia coli K4 fermentation broth was developed. The process is simple, scalable and economical. In particular, downstream procedures were optimized with a particular aim of purifying a product suitable for further chemical modifications, in an attempt to develop a biotechnological platform for chondroitin sulfate production. During process development, membrane devices (ultrafiltration/diafiltration) were exploited, selecting the right cassette cut-offs for different phases of purification. The operational conditions (cross-flow rate and transmembrane pressure) used for the process were determined on an ?KTA cross-flow instrument (GE Healthcare, USA), a lab-scale automatic tangential flow filtration system. In addition, parameters such as selectivity and throughput were calculated based on the analytical quantification of K4 and defructosylated K4, as well as the major contaminants. The complete downstream procedure yielded about 75% chondroitin with a purity higher than 90%.     

Purification of plasmid DNA from Escherichia coli ferments using anion-exchange membrane and hydrophobic chromatography

A novel downstream bioprocess was developed to obtain purified plasmid DNA (pDNA) from Escherichia coli ferments. The intermediate recovery and purification of the pDNA in cell lysate was conducted using hollow-fiber tangential filtration and frontal anion-exchange membrane and elution hydrophobic chromatographies. The purity of the solutions of pDNA obtained during each process stage was investigated. The results show that the pDNA solution purity increased 30-fold and more than 99% of RNA in the lysate was removed during the process operations. The combination of membrane operations and hydrophobic interaction chromatography resulted in an efficient way to recover pDNA from cell lysates. A better understanding of membrane-based technology for the purification of pDNA from clarified E. coli lysate was developed in this research.     

Structural characterization of the O-chain polysaccharide from an environmentally beneficial bacterium Pseudomonas chlororaphis subsp. aureofaciens strain M71

Pseudomonas chlororaphis subsp. aureofaciens strain M71 was isolated from the root of a tomato plant and it was able to control in vivo Fusarium oxysporum f. sp. radicis-lycopersici responsible for the tomato crown and root rot. Recently, strain M71 was evaluated even for its efficacy in controlling Seiridium cardinale, the causal agent of bark canker of common cypress (Cupressus sempervirens L.). Strain M71 ability to persist on the tomato rhizosphere and on the aerial part of cypress plants could be related to the nature of the lipopolysaccharides (LPS) present on the outer membrane and in particular to the O-specific polysaccharide.A neutral O-specific polysaccharide was obtained by mild acid hydrolysis of the lipopolysaccharide from P. chlororaphis subsp. aureofaciens strain M71. By means of compositional analyses and NMR spectroscopy, the chemical repeating unit of the polymer was identified as the following linear trisaccharide.     

Preliminary characterization and grouping of Candida species by numerical analysis of protein profiles obtained by polyacrylamide gel electrophoresis

Whole-cell proteins from isolates of five Candida species (Candida albicans, Candida krusei, Candida parapsilosis, Candida tropicalis and Candida guilliermondii) were separated by SDS-PAGE and the profiles obtained were converted into a binary data matrix that produced a cophenetic correlation phenogram. The analysis of the phenogram allowed detection of the cophenetic correlation levels existing among these species.     

SKP2 oncogene is a direct MYC target gene and MYC down-regulates p27(KIP1) through SKP2 in human leukemia cells

SKP2 is the ubiquitin ligase subunit that targets p27(KIP1) (p27) for degradation. SKP2 is induced in the G(1)-S transit of the cell cycle, is frequently overexpressed in human cancer, and displays transformation activity in experimental models. Here we show that MYC induces SKP2 expression at the mRNA and protein levels in human myeloid leukemia K562 cells with conditional MYC expression. Importantly, in these systems, induction of MYC did not activate cell proliferation, ruling out SKP2 up-regulation as a consequence of cell cycle entry. MYC-dependent SKP2 expression was also detected in other cell types such as lymphoid, fibroblastic, and epithelial cell lines. MYC induced SKP2 mRNA expression in the absence of protein synthesis and activated the SKP2 promoter in luciferase reporter assays. With chromatin immunoprecipitation assays, MYC was detected bound to a region of human SKP2 gene promoter that includes E-boxes. The K562 cell line derives from human chronic myeloid leukemia. In a cohort of chronic myeloid leukemia bone marrow samples, we found a correlation between MYC and SKP2 mRNA levels. Analysis of cancer expression databases also indicated a correlation between MYC and SKP2 expression in lymphoma. Finally, MYC-induced SKP2 expression resulted in a decrease in p27 protein in K562 cells. Moreover, silencing of SKP2 abrogated the MYC-mediated down-regulation of p27. Our data show that SKP2 is a direct MYC target gene and that MYC-mediated SKP2 induction leads to reduced p27 levels. The results suggest the induction of SKP2 oncogene as a new mechanism for MYC-dependent transformation.     

In vitro cytotoxic activity of extracts and isolated constituents of Salvia leriifolia Benth. against a panel of human cancer cell lines

In the course of recent efforts to identify new potential antiproliferative active principles, Salvia leriifolia extracts and isolated constituents were evaluated for their cytotoxic activity against a panel of human cancer cell lines, including renal adenocarcinoma (ACHN), amelanotic melanoma (C32), colorectal adenocarcinoma (Caco‐2), lung large cell carcinoma (COR‐L23), malignant melanoma (A375), lung carcinoma (A549), and hepatocellular carcinoma (Huh‐7D12) cells. The hexane and CH₂Cl₂ extracts showed the strongest cytotoxic activity against the C32 cell line with IC₅₀ values of 11.2 and 13.6 μg/ml, respectively, and the AcOEt extract was the most active extract against the COR‐L23 cell line (IC₅₀ of 20.9 μg/ml). Buchariol, a sesquiterpene obtained by biofractionation of the CH₂Cl₂ extract, exhibited a higher activity than the positive control vinblastine against the C32 and A549 cell lines (IC₅₀ values of 2.1 and 12.6 μM , resp.). Interesting results were also obtained for naringenin, a flavonoid isolated from the AcOEt extract, which exhibited a strong cytotoxic activity against the C32, LNCaP, and COR‐L23 cell lines (IC₅₀ values of 2.2, 7.7, and 33.4 μM , resp.), compared to vinblastine (IC₅₀ values of 3.3, 32.2, 50.0 μM , resp.). None of the tested compounds affected the proliferation of skin fibroblasts (142BR), suggesting a selective activity against tumor cells.     

Protein polymorphism inEligmodontia typus. Genetic divergence with other phyllotine cricetids

By means of gel electrophoresis of tissue extracts we have studied protein polymorphism inEligmodontia typus. The analysis was performed on specimens from five population samples collected at different sites in Patagonia (Argentina). Mean heterozygosity (\-h) and proportion of polymorphic loci (P) were determined on the basis of 19 loci. Considering all individuals as one sample, \-h gave a value of 0.16 and P was 70%. Although these values are much higher than those reported for most rodent species, they are very similar to those obtained by us for four species of the genusCalomys and forGraomys griseoflavus. There is a striking genetic identity (I_N=0.99) among populations from regions with different environmental conditions, indicating that the species possesses a common genic pool. Genetic distance with other species of the Phyllotini was estimated. D_N was lower betweenE. typus andCalomys (mean D_N=0.88) than betweenE. typus andGraomys griseoflavus (D_N=1.01). The high morphological similarity between these last two species, especially regarding those characters related to desert life adaptation, could be assigned, at least in part, to convergent evolution.     

Déficit de vitamina D en una cohorte de mayores de 65 años: prevalencia y asociación con factores sociodemográficos y de salud

Introduction

Vitamin D deficiency is common in the elderly, especially among institutionalized and/or hip fracture patients. However, there are few population studies on the prevalence of this deficiency in the general population over 64 years in our environment. The aim of this study was to determine the prevalence of vitamin D deficiency in an urban population cohort of over 64 years, and analyze its relationship with sociodemographic, climatic, and health factors.

Material and methods

Cross-sectional study from «Peñagrande cohort», a population-based cohort consisting of people over 64 years. We determined 25-hydroxyvitamin D levels, and recorded sociodemographic data (age, sex, marital status, education, socioeconomic status), season of measurement and health variables (comorbidity, obesity, malnutrition, renal failure, cognitive impairment, vitamin D supplements, and disability).

Results

A total of 468 individuals with a mean age of 76.0 years (SD: 7.7) were included, of which 53.4% were women. The mean value of vitamin D was 20.3 ± 11.7 ng/mL. The large majority (86.3%, 95% CI: 83.0-89.5) had a vitamin insufficiency (≤ 30 ng/ml), and 35.2% (95% CI: 30.8-39.7) showed severe vitamin deficiency (≤ 15 ng/ml). Vitamin insufficiency increases linearly with age (OR 1.06; 95% CI: 1.01-1.11), and was associated with low socioeconomic status (OR 3.29; 95% CI: 1.55-6.95). Severe vitamin D deficiency increases with age (OR 1.06; 95% CI: 1.02-1.09), female gender (OR 1.80; 95% CI: 1.18-2.75) and with cognitive impairment (OR 1.71; 95% CI: 1.04-2.83).

Conclusion

The prevalence of vitamin D deficiency in people over 65 years of age in our community is high. It would be advisable to determine the vitamin D values in the high risk elderly in order to introduce measures of pharmacological supplementation in those with inadequate levels.