A quantitative method to evaluate mesenchymal stem cell lipofection using real‐time PCR

Genetic modification of human mesenchymal stem cells (MSC) is a powerful tool to improve the therapeutic utility of these cells and to increase the knowledge on their regulation mechanisms. In this context, strong efforts have been made recently to develop efficient nonviral gene delivery systems. Although several studies addressed this question most of them use the end product of a reporter gene instead of the DNA uptake quantification to test the transfection efficiency. In this study, we established a method based on quantitative real‐time PCR (RT‐PCR) to determine the intracellular plasmid DNA copy number in human MSC after lipofection. The procedure requires neither specific cell lysis nor DNA purification. The influence of cell number on the RT‐PCR sensitivity was evaluated. The method showed good reproducibility, high sensitivity, and a wide linear range of 75–2.5 × 10⁶ plasmid DNA copies per cell. RT‐PCR results were then compared with the percentage of transfected cells assessed by flow cytometry analysis, which showed that flow cytometry‐based results are not always proportional to plasmid cellular uptake determined by RT‐PCR. This work contributed for the establishment of a rapid quantitative assay to determine intracellular plasmid DNA in stem cells, which will be extremely beneficial for the optimization of gene delivery strategies. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Partition of alpha-lactoalbumin and beta-lactoglobulin by cloud point extraction

This work aimed to study the partition of cheese whey proteins alpha-lactoalbumin and beta-lactoglobulin using aqueous two-phase system by applying the cloud point extraction technique. The cloud point temperatures were determined under different concentrations of copolymer and salt. The system providing the best protein separation conditions was 20 mass% of copolymer PE61 and potassium phosphate salt solution of 100 mM, at pH 7. The protein alpha-lactoalbumin remained preferentially in the aqueous phase and the beta-lactoglobulin was transferred to the copolymer phase.     

Reproductive steroid hormones and ovarian activity in felids of the Leopardus genus

Reproductive endocrine patterns were characterized in female ocelots (Leopardus pardalis; n = 3), tigrinas (Leopardus tigrinus; n = 2), and margays (Leopardus wiedii; n = 2) housed in captivity in southern Brazil. Females were maintained as singletons and exposed to natural fluctuations in photoperiod. Cyclic changes in ovarian steroids were monitored by analyzing estrogen and progestogen metabolites in fecal samples collected five times weekly for 14 to 18 months. Based on intervals between fecal estrogen peaks, mean (± SEM) duration of the estrous cycle was 18.4 ± 1.6 days for the ocelots (range, 7–31 days; n = 75 cycles), 16.7 ± 1.3 days for the tigrinas (range, 11–27 days; n = 23 cycles), and 17.6 ± 1.5 days for the margays (range, 11–25 days; n = 32 cycles). Fecal progestogen analyses combined with two laparoscopic observations of the ovaries confirmed that ocelots and tigrinas did not ovulate spontaneously. In contrast, non‐mating–induced luteal phases of 40.1 ± 6.3 days in duration (range, 30–60 days) were observed frequently in both margays. There was no evidence of gonadal seasonality in margays in either follicular or luteal activity. In ocelots, cyclic changes in estrogen excretion were observed during each month of the year; however, only one female cycled continuously. In the other two ocelots, periods of acyclicity of several months’ duration were observed. It was not possible to conclude whether tigrinas were aseasonal because estrous cyclicity was observed in only one of two individuals. In the female that cycled, a 3‐month period of acyclicity was observed in the late fall/early winter. These data demonstrate similarities among three felid species of the genus Leopardus, including evidence they are polyestrous but experience unexplained periods of ovarian inactivity. Only the margays differed by exhibiting occasional spontaneous, non‐mating–induced ovulations. Historically, these species have not bred well in captivity. However, it is hoped that understanding the biological similarities and differences among them could lead to improved management strategies that may one day result in increased reproductive success. Zoo Biol 20:103–116, 2001. © 2001 Wiley‐Liss, Inc.     

Helicobacter pylori from asymptomatic hosts expressing heptoglycan but lacking Lewis O-chains: Lewis blood-group O-chains may play a role in Helicobacter pylori induced pathology.

Helicobacter pylori is a widespread Gram-negative bacterium responsible for the onset of various gastric pathologies and cancers in humans. A familiar trait of H. pylori is the production of cell-surface lipopolysaccharides (LPSs; O-chain --> core --> lipid A) with O-chain structures analogous to some mammalian histo-blood-group antigens, those being the Lewis determinants (Lea, Leb, Lex, sialyl Lex, Ley) and blood groups A and linear B. Some of these LPS antigens have been implicated as autoimmune, adhesion, and colonization components of H. pylori pathogenic mechanisms. This article describes the chemical structures of LPSs from H. pylori isolated from subjects with no overt signs of disease. Experimental data from chemical- and spectroscopic-based studies unanimously showed that these H. pylori manufactured extended heptoglycans composed of 2- and 3-linked D-glycero-alpha-D-manno-heptopyranose units and did not express any blood-group O-antigen chains. The fact that another H. pylori isolate with a similar LPS structure was shown to be capable of colonizing mice indicates that H. pylori histo-blood-group structures are not an absolute prerequisite for colonization in the murine model also. The absence of O-chains with histo-blood groups may cause H. pylori to become inept in exciting an immune response. Additionally, the presence of elongated heptoglycans may impede exposure of disease-causing outer-membrane antigens. These factors may render such H. pylori incapable of creating exogenous contacts essential for pathogenesis of severe gastroduodenal diseases and suggest that histo-blood groups in the LPS may indeed play a role in inducing a more severe H. pylori pathology.     

On-Line Detection of Microbial Contaminations in Animal Cell Reactor Cultures Using an Electronic Nose Device

An electronic nose (EN) device was used to detect microbial and viral contaminations in a variety of animal cell culture systems. The emission of volatile components from the cultures accumulated in the bioreactor headspace, was sampled and subsequently analysed by the EN device. The EN, which was equipped with an array of 17 chemical gas sensors of varying selectivity towards the sampled volatile molecules, generated response patterns of up to 85 computed signals. Each 15 or 20 min a new gas sample was taken generating a new response pattern. A software evaluation tool visualised the data mainly by using principal component analysis. The EN was first used to detect microbial contaminations in a Chinese hamster ovary (CHO) cell line producing a recombinant human macrophage colony stimulating factor (rhM-CSF). The CHO cell culture was contaminated by Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Candida utilis which all were detected. The response patterns from the CHO cell culture were compared with monoculture references of the microorganisms. Second, contaminations were studied in an Sf-9 insect cell culture producing another recombinant protein (VP2 protein). Contaminants were detected from E. coli, a filamentous fungus and a baculovirus. Third, contamination of a human cell line, HEK-293, infected with E. coli exhibited comparable results. Fourth, bacterial contaminations could also be detected in cultures of a MLV vector producer cell line. Based on the overall experiences in this study it is concluded that the EN method has in a number of cases the potential to be developed into a useful on-line contamination alarm in order to support safety and economical operation for industrial cultivation.     

Sulfated galactofucan from <Emphasis Type="Italic">Lobophora variegata</Emphasis>: Anticoagulant and anti-inflammatory properties

Sulfated polysaccharides (fucans and fucoidans) from brown algae show several biological activities, including anticoagulant and anti-inflammatory activities. We have extracted a sulfated heterofucan from the brown seaweed Lobophora variegata by proteolytic digestion, followed by acetone fractionation, molecular sieving, and ion-exchange chromatography. Chemical analyses and ¹³C-NMR and IR spectroscopy showed that this fucoidan is composed of fucose, galactose, and sulfate at molar ratios of 1:3:2. We compared the anticoagulant activity of L. variegata fucoidan with those of a commercial sulfated polysaccharide (also named fucoidan) from Fucus vesiculosus and heparin. The experimental inflammation models utilized in this work revealed that fucoidan from L. variegata inhibits leukocyte migration to the inflammation site. Ear swelling caused by croton oil was also inhibited when sulfated polysaccharides from F. vesiculosus and L. variegata were used. The precise mechanism of different action between homo-and heterofucans is not clear; nevertheless, the polysaccharides studied here may have therapeutic potential in inflammatory disorders. Published in Russian in Biokhimiya, 2008, Vol. 73, No. 9, pp. 1265–1273.     

Sulfated galactan is a catalyst of antithrombin-mediated inactivation of alpha-thrombin

Novel compounds presenting anticoagulant activity, such as sulfated polysaccharides, open new perspectives in medicine. Elucidation of the molecular mechanism behind this activity is desirable by itself, as well as because it allows for the design of novel compounds. In the present study, we investigated the action of an algal sulfated galactan, which potentiates alpha-thrombin inactivation by antithrombin. Our results indicate the following: 1) both the sulfated galactan and heparin potentiate protease inactivation by antithrombin at similar molar concentrations, however they differ markedly in the molecular size required for their activities; 2) this galactan interacts predominantly with exosite II on alpha-thrombin and, similar to heparin, catalyzes the formation of a covalent complex between antithrombin and the protease; 3) the sulfated galactan has a higher affinity for alpha-thrombin than for antithrombin. We propose that the preferred pathway of sulfated galactan-induced inactivation of alpha-thrombin by antithrombin starts with the polysaccharide binding to the protease through a high-affinity interaction. Antithrombin is then added to the complex and the protease is inactivated by covalent interactions. Finally, the antithrombin-alpha-thrombin covalent complex dissociates from the polysaccharide chain. This mechanism resembles the action of heparin with low affinity for antithrombin, as opposed to heparin with high affinity for serpin.     

New insights into conformational and functional stability of human alpha-thrombin probed by high hydrostatic pressure.

The effects of high hydrostatic pressure (HHP) and urea on conformational transitions of human alpha-thrombin structure were studied by fluorescence spectroscopy and by measuring the catalytic activity of the enzyme. Treatment of thrombin with urea produced a progressive red shift in the center of mass of the intrinsic fluorescence emission spectrum, with a maximum displacement of 650 cm(-1). HHP (270 MPa) shifted the centre of mass by only 370 cm(-1). HHP combined with a subdenaturing urea concentration (1.5 m) displaced the centre of mass by approximately 750 cm(-1). The binding of the fluorescent probe bis(8-anilinonaphthalene-1-sulfonate) to thrombin was increased by 1.8-, 4.0-, and 2.7-fold after treatment with high urea concentration, HHP or HHP combined with urea, respectively, thus suggesting that all treatments convert the enzyme to partially folded intermediates with exposed hydrophobic regions. On the other hand, treatment of thrombin with urea (but not HHP) combined with dithiothreitol progressively displaced the fluorescent probe, thus suggesting that this condition converts the enzyme to a completely unfolded state. Urea and HHP also led to different conformations when changes in the thrombin catalytic site environment were assessed using the fluorescence emission of fluorescein-d-Phe-Pro-Arg-cloromethylketone-alpha-thrombin: addition of urea up to 2 m gradually decreased the fluorescence emission of the probe to 65% of the initial intensity, whereas HHP caused a progressive increase in fluorescence. Hydrolysis of the synthetic substrate S-2238 was enhanced (35%) in 2 m urea and gradually abolished at higher concentrations, while HHP (270 MPa) inhibited the enzyme's catalytic activity by 45% and abolished it when 1.5 m urea was also present. Altogether, analysis of urea and HHP effects on thrombin structure and activity indicates the formation of dissimilar intermediate states during denaturation by these agents.