Calfection: a novel gene transfer method for suspension cells

We have developed a novel method called Calfection for gene delivery to and protein expression from suspension-cultivated mammalian cells. Plasmid DNA was simply diluted into a calcium chloride solution and then added to the cell culture for transfection. We evaluated and optimized this approach using suspension-adapted HEK293 cells grown in 12-well plates that were shaken on an orbital shaker. Highest expression levels were obtained when cells were transfected at a density of 5x10(5) cells/ml in the presence of 9 mM calcium and 5 microg/ml of plasmid DNA while maintaining a culture pH of 7.6 at the time of transfection. Suspension-adapted BHK 21 and CHO DG 44 cells could also be transfected using this method. Calfection differs from the widely known calcium phosphate coprecipitation technique. The physico-chemical composition of the DNA interacting complexes is not yet known. The transfection cocktail, DNA in a calcium chloride solution, remained highly efficient during long-term storage at temperatures ranging from room temperature to -80 degrees C. In contrast, calcium phosphate-DNA cocktails are only efficient for gene transfer when prepared fresh. Furthermore, passing the calcium-plasmid DNA mixture through a 0.2-microm filter did not compromise protein expression, whereas calcium phosphate-DNA coprecipitates were retained by the filter. High protein expression levels, a limited number of manipulations and the possibility to filter the cocktail make the Calfection approach suitable for both large-scale transfection in bioreactors and for high-throughput transfection experiments in microtiter plates.     

Molecular interference of Cd(2+) with Photosystem II

Many heavy metals inhibit electron transfer reactions in Photosystem II (PSII). Cd(2+) is known to exchange, with high affinity in a slow reaction, for the Ca(2+) cofactor in the Ca/Mn cluster that constitutes the oxygen-evolving center. This results in inhibition of photosynthetic oxygen evolution. There are also indications that Cd(2+) binds to other sites in PSII, potentially to proton channels in analogy to heavy metal binding in photosynthetic reaction centers from purple bacteria. In search for the effects of Cd(2+)-binding to those sites, we have studied how Cd(2+) affects electron transfer reactions in PSII after short incubation times and in sites, which interact with Cd(2+) with low affinity. Overall electron transfer and partial electron transfer were studied by a combination of EPR spectroscopy of individual redox components, flash-induced variable fluorescence and steady state oxygen evolution measurements. Several effects of Cd(2+) were observed: (i) the amplitude of the flash-induced variable fluorescence was lost indicating that electron transfer from Y(Z) to P(680)(+) was inhibited; (ii) Q(A)(-) to Q(B) electron transfer was slowed down; (iii) the S(2) state multiline EPR signal was not observable; (iv) steady state oxygen evolution was inhibited in both a high-affinity and a low-affinity site; (v) the spectral shape of the EPR signal from Q(A)(-)Fe(2+) was modified but its amplitude was not sensitive to the presence of Cd(2+). In addition, the presence of both Ca(2+) and DCMU abolished Cd(2+)-induced effects partially and in different sites. The number of sites for Cd(2+) binding and the possible nature of these sites are discussed.     

Lipid dependence and activity control of phosphatidylserine synthase from Escherichia coli

The activity of phosphatidylserine synthase from Escherichia coli depends significantly on the nature and level of the lipids in the matrix, at which the enzyme is operating. To elucidate the role of anionic lipids in the regulation of PtdSer synthase, its activity was studied in mixed micelles containing phosphatidylglycerol (one charge) or diphosphatidylglycerol (two charges), the two main anionic membrane lipids in E. coli. Membrane association and activity of PtdSer synthase were increased by the two lipids, indicating their essential role in the positive regulation mechanism of the phosphatidylethanolamine level in the E. coli membrane.     

Height Changes Associated with Pigment Aggregation in <Emphasis Type="Italic">Xenopus laevis</Emphasis> Melanophores

Melanophores are pigment cells found in the skin of lower vertebrates. The brownish-black pigment melanin is stored in organelles called melanosomes. In response to different stimuli, the cells can redistribute the melanosomes, and thereby change colour. During melanosome aggregation, a height increase has been observed in fish and frog melanophores across the cell centre. The mechanism by which the cell increases its height is unknown. Changes in cell shape can alter the electrical properties of the cell, and thereby be detected in impedance measurements. We have in earlier studies of Xenopus laevis melanophores shown that pigment aggregation can be revealed as impedance changes, and therefore we were interested in investigating the height changes associated with pigment aggregation further. Accordingly, we quantified the changes in cell height by performing vertical sectioning with confocal microscopy. In analogy with theories explaining the leading edge of migrating cells, we investigated the possibility that the elevation of plasma membrane is caused by local swelling due to influx of water through HgC1₂-sensitive aquaporins. We also measured the height of the microtubule structures to assess whether they are involved in the height increase. Our results show that pigment aggregation in X. laevis melanophores resulted in a significant height increase, which was substantially larger when aggregation was induced by latrunculin than with melatonin. Moreover, the elevation of the plasma membrane did not correlate with influx of water through aquaporins or formation of new microtubules, Rather, the accumulation of granules seemed to drive the change in cell height.     

Stability of peptide-HLA-I complexes and tapasin folding facilitation--tools to define immunogenic peptides

Only a small fraction of the peptides generated inside the cell end up being presented by HLA-I on the cell surface. High stability of peptide–HLA-I complexes and a low HLA-I tapasin-facilitation have been proposed to predict immunogenicity. We here set out to investigate if these parameters correlated and defined immunogenic peptides. Both peptide–HLA–B¹08:01 and peptide–HLA–A¹02:01 complexes showed small differences in tapasin-facilitation and larger differences in stability. This suggests that the stability of immunogenic peptide–HLA-I complexes vary above an HLA-I allomorph dependent lower limit (e.g. >2 h for HLA–A¹02:01), immunogenicity predicted by tapasin-facilitation may be defined by an equally allomorph unique upper value (e.g. tapasin-facilitation <1.5 for HLA–A¹02:01), and variation above the stability-threshold does not directly reflect a variation in tapasin-facilitation.     

The P-SSP7 cyanophage has a linear genome with direct terminal repeats

P-SSP7 is a T7-like phage that infects the cyanobacterium Prochlorococcus MED4. MED4 is a member of the high-light-adapted Prochlorococcus ecotypes that are abundant in the surface oceans and contribute significantly to primary production. P-SSP7 has become a model system for the investigation of T7-like phages that infect Prochlorococcus. It was classified as T7-like based on genome content and organization. However, because its genome assembled as a circular molecule, it was thought to be circularly permuted and to lack the direct terminal repeats found in other T7-like phages. Here we sequenced the ends of the P-SSP7 genome and found that the genome map is linear and contains a 206 bp repeat at both genome ends. Furthermore, we found that a 728 bp region of the genome originally placed downstream of the last ORF is actually located upstream of the first ORF on the genome map. These findings suggest that P-SSP7 is likely to use the direct terminal repeats for genome replication and packaging in a similar manner to other T7-like phages. Moreover, these results highlight the importance of experimentally verifying the ends of phage genomes, and will facilitate the use of P-SSP7 as a model for the correct assembly and end determination of the many T7-like phages isolated from the marine environment that are currently being sequenced.     

Lipopolysaccharide O-antigen prevents phagocytosis of Vibrio anguillarum by rainbow trout (Oncorhynchus mykiss) skin epithelial cells

Colonization of host tissues is a first step taken by many pathogens during the initial stages of infection. Despite the impact of bacterial disease on wild and farmed fish, only a few direct studies have characterized bacterial factors required for colonization of fish tissues. In this study, using live-cell and confocal microscopy, rainbow trout skin epithelial cells, the main structural component of the skin epidermis, were demonstrated to phagocytize bacteria. Mutant analyses showed that the fish pathogen Vibrio anguillarum required the lipopolysaccharide O-antigen to evade phagocytosis and that O-antigen transport required the putative wzm-wzt-wbhA operon, which encodes two ABC polysaccharide transporter proteins and a methyltransferase. Pretreatment of the epithelial cells with mannose prevented phagocytosis of V. anguillarum suggesting that a mannose receptor is involved in the uptake process. In addition, the O-antigen transport mutants could not colonize the skin but they did colonize the intestines of rainbow trout. The O-antigen polysaccharides were also shown to aid resistance to the antimicrobial factors, lysozyme and polymyxin B. In summary, rainbow trout skin epithelial cells play a role in the fish innate immunity by clearing bacteria from the skin epidermis. In defense, V. anguillarum utilizes O-antigen polysaccharides to evade phagocytosis by the epithelial cells allowing it to colonize rapidly fish skin tissues.     

Short-term psychological outcomes in severely obese adolescents after bariatric surgery

Bariatric surgery is suggested as a treatment option for severely obese adolescents. Because adolescence is characterized by intense psychosocial adjustment and development, it is important to study the effect of this procedure on adolescents' psychological health. This study examined baseline status and short-term changes in anxiety, depression, anger, disruptive behavior, and self-concept in 37 adolescents (mean age 16.6 ± 1.3). Participants completed the Beck Youth Inventories (BYI) at inclusion and (on average) 4 months after undergoing Roux-en-Y gastric bypass (RYGB). Internalizing (anxiety and depression) and externalizing (anger and disruptive behavior) symptoms were higher at baseline than gender-specific norms. One fifth had a very low self-concept. Four months after surgery, the adolescents showed significantly fewer symptoms of anxiety and depression and significantly improved self-concept from baseline. Anger and disruptive behavior showed no significant changes. An analysis of clinically meaningful changes was conducted, and besides the overall positive outcome, 16% (n = 6) of the adolescents had deteriorated on two or more inventories in BYI shortly after surgery. This impaired group did not show any specific features at inclusion. The results indicate the importance of psychological monitoring immediately after bariatric surgery and the need for additional psychosocial support to be available for vulnerable sub-groups of adolescents. Further studies with larger samples are necessary to identify characteristics predictive of short-term adverse psychological outcomes in adolescents after bariatric surgery.     

ITIH-5 expression in human adipose tissue is increased in obesity

Adipocytes secrete many proteins that regulate metabolic functions. The gene inter-α (globulin) inhibitor H5 (ITIH-5) encodes a secreted protein and is known to be expressed abundantly in the placenta. However, using gene expression profiles data we observed high expression of ITIH-5 in adipose tissue. The aim of this study was to test the hypothesis that ITIH-5 is strongly expressed in human adipocytes and adipose tissue, and is related to obesity and clinical metabolic variables. ITIH-5 adipose tissue mRNA expression was analyzed with DNA microarray and real-time PCR, and its association with clinical variables was examined. ITIH-5 protein expression was analyzed using western blot. ITIH-5 mRNA expression was abundant in human adipose tissue, adipocytes, and placenta, and higher in subcutaneous (sc) compared to omental adipose tissue (P < 0.0001). ITIH-5 mRNA and protein expression in sc adipose tissue were higher in obese compared to lean subjects (P < 0.0001 and P < 0.001, respectively). ITIH-5 mRNA expression was reduced after diet-induced weight loss (P < 0.0001). ITIH-5 mRNA expression was associated with anthropometry and clinical metabolic variables. In conclusion, ITIH-5 is highly expressed in sc adipose tissue, increased in obesity, down regulated after weight loss, and associated with measures of body size and metabolism. Together, this indicates that ITIH-5 merits further investigation as a regulator of human metabolism.     

Cleavage Mediated by the Catalytic Domain of Bacterial RNase P RNA

Like other RNA molecules, RNase P RNA (RPR) is composed of domains, and these have different functions. Here, we provide data demonstrating that the catalytic (C) domain of Escherichia coli (Eco) RPR when separated from the specificity (S) domain mediates cleavage using various model RNA hairpin loop substrates. Compared to full-length Eco RPR, the rate constant, k(obs), of cleavage for the truncated RPR (CP RPR) was reduced 30- to 13,000-fold depending on substrate. Specifically, the structural architecture of the -1/+73 played a significant role where a C(-1)/G(+73) pair had the most dramatic effect on k(obs). Substitution of A(248) (E. coli numbering), positioned near the cleavage site in the RNase P-substrate complex, with G in the CP RPR resulted in 30-fold improvement in rate. In contrast, strengthening the interaction between the RPR and the 3' end of the substrate only had a modest effect. Interestingly, although deleting the S-domain gave a reduction in the rate, it resulted in a less erroneous RPR with respect to cleavage site selection. These data support and extend our understanding of the coupling between the distal interaction between the S-domain and events at the active site. Our findings will also be discussed with respect to the structure of RPR derived from different organisms.