Prediction of affinity and kinetics in biomolecular interactions by affinity chromatography

Computer simulation of affinity chromatography is a valuable tool for accurate prediction of column performance. In our study affinity pairs based on lectin and antibody interactions with carbohydrates have been used as model systems. In this well-characterized system we have demonstrated the usefulness of the simulation approach for determination of affinity and kinetics. These properties are typically difficult to obtain for many weakly interacting molecular species (i.e., when dissociation constants (K(D)) are greater than 10(-5) M). The influence of affinity and kinetics on peak broadening in affinity chromatography has also been investigated.     

Microsatellite markers for Hylocomium splendens (Hylocomiaceae)

? Premise of the study: The perennial feather moss Hylocomium splendens is one of the most widely distributed and common bryophytes in the Northern Hemisphere and has, because of its capacity to grow under a wide range of environmental conditions, been used as a biomonitor for atmospheric metal deposition in Europe. ? Methods and Results: We present a multiplex approach for the analysis of 14 microsatellite markers tested on 194 H. splendens gametophytes. Ten of the markers are developed recently, and are presented for the first time in this paper, whereas four were previously developed but have not been used for population genetic investigations. ? Conclusions: The microsatellite markers reported here will provide a powerful tool for further research on population genetic structure in H. splendens.     

Site identity and moss species as determinants of soil microbial community structure in Norway spruce forests across three vegetation zones

Soil microbial community structure was investigated by PLFA-analysis in four spruce forests in Norway. The maximum latitudinal distance between the sites was approximately 350 km. Bilberry Vaccinium myrtillus dominated the forest floor vegetation in the study sites, which were selected because of the vegetation type. Soil samples were taken from all four sites under close to 100% homogeneous ground cover of each of two feathermoss species, i.e. Hylocomium splendens or Pleurozium schreberi, respectively. These mosses are ubiquitous in the boreal forest and constitute an abundant component of the forest floor vegetation over vast areas. Since there are no studies on how these mosses affect soil microbial community structure, our first aim was to investigate the effect of moss species on soil microbial communities. Our second aim was to investigate whether microbial communities differ among geographically separated forest sites with similar vegetation across vegetation zones. Soil microbial community structure differed between the study sites, although they appeared similar in terms of vegetation and abiotic soil conditions. Study site was the most important predictor of the variation in the PLFAs, more important than moss species, although there was a tendency for separation of microbial community structure between the two moss species.     

Weak affinity chromatography as a new approach for fragment screening in drug discovery

Fragment-based drug design (FBDD) is currently being implemented in drug discovery, creating a demand for developing efficient techniques for fragment screening. Due to the intrinsic weak or transient binding of fragments (mM–μM in dissociation constant (K_D)) to targets, methods must be sensitive enough to accurately detect and quantify an interaction. This study presents weak affinity chromatography (WAC) as an alternative tool for screening of small fragments. The technology was demonstrated by screening of a selected 23-compound fragment collection of documented binders, mostly amidines, using trypsin and thrombin as model target protease proteins. WAC was proven to be a sensitive, robust, and reproducible technique that also provides information about affinity of a fragment in the range of 1 mM–10 μM. Furthermore, it has potential for high throughput as was evidenced by analyzing mixtures in the range of 10 substances by WAC–MS. The accessibility and flexibility of the technology were shown as fragment screening can be performed on standard HPLC equipment. The technology can further be miniaturized and adapted to the requirements of affinity ranges of the fragment library. All these features of WAC make it a potential method in drug discovery for fragment screening.     

Structure and function of Salmonella SifA indicate that its interactions with SKIP, SseJ, and RhoA family GTPases induce endosomal tubulation

The Salmonella typhimurium type III secretion effector protein SifA is essential for inducing tubulation of the Salmonella phagosome and binds the mammalian kinesin-binding protein SKIP. Coexpression of SifA with the effector SseJ induced tubulation of mammalian cell endosomes, similar to that induced by Salmonella infection. Interestingly, GTP-bound RhoA, RhoB, and RhoC also induced endosomal tubulation when coexpressed with SseJ, indicating that SifA likely mimics or activates a RhoA family GTPase. The structure of SifA in complex with the PH domain of SKIP revealed that SifA has two distinct domains; the amino terminus binds SKIP, and the carboxyl terminus has a fold similar to SopE, a Salmonella effector with Rho GTPase guanine nucleotide exchange factor activity (GEF). Similar to GEFs, SifA interacted with GDP-bound RhoA, and purified SseJ and RhoA formed a protein complex, suggesting that SifA, SKIP, SseJ, and RhoA family GTPases cooperatively promote host membrane tubulation.     

Variation in tissue element concentration in mire plants over a range of sites

Elements in surface peat and ground water were estimated on seven mire sites in central and north Sweden. The variation in tissue clement concentration was studied in eleven plants occurring on the studied sites. The elements estimated were: N, P, K. Ca, Mg, Fe, Mn, Zn, Al, Cu, Mo, Na, S, B, and Si. The species studied were; Carex diandra, Carex limosa, Carex rostrata, Epilobium palustre, Menyanthes trifoliate, Pedicularis palustris, Rumex acetosa, Saxifraga hirculus, Scirpus hudsonianus, Stellaria crassifolia and Tofteldia pusilla . In surface peat over the range of sites, the variation in total concentrations of nitrogen and phosphorus was six and tenfold, respectively. The variation in total concentration of potassium was four-fold. Extractable concentrations of these three elements showed an even greater variation between sites. Over the range of sites, the variation in tissue element concentration was larger in roots than in above-ground parls. In above-ground parts, floral parts varied less in tissue element concentration than did stems and leaves. The largest variation in tissue element concentration was found in Carex rostrata and Menyanthes trifoliata . The smallest variation was found in Saxifraga hirculus . Tissues of the rich fen species Saxifraga hirculus and Stellaria crassifolia had the lowest concentrations of heavy metals. The correlation between substrate element concentration and tissue concentration differed both between tissues and species. Concentrations of N, P and K in tissues of the Carex species were highly correlated with the concentrations of these elements in the substrate, while N, P and K concentrations in tissues of the herbaceous species, especially Saxifraga hirculus . correlated less well.     

Reproductive differentiation in a Saxifraga hirculus population along an environmental gradient on a central Swedish mire

The perennial herbaceous mire plant Saxifraga hirculus was studied in different population densities along an environmental gradient on a mire in central Sweden. Tests were made whether the habitat conditions influenced the balance between vegetative growth of runners and seed production. Along a 30 m long transect the gradient was analysed for vegetation, hydrology, temperature and contents of ions in groundwater and surface peat. Three main zones were distinguished, a spring area, a rich fen area and an intermediate fen area. Range in concentration of ions in groundwater along the transect (mg 1⁻¹): Ca 4–59, Mg 0.3–3.5, K 0.1–3.0, Na 1.0–4.1 and Fe 0.4–4.5. Range in pH: 5.8–7.5. Despite the gradient in ion concentration, the element content in S. hirculus was constant along the transect. Population density reached maximum in the rich fen, 52 floral shoots 0.25 m⁻². In the spring area, S. hirculus was characterized by few flowers per floral shoot, low height, numerous runners and a large total length of runners. In the rich fen, high seed production was characteristic; in the intermediate fen, production of both runners and seeds were low. The flowering of S. hirculus starts early in the spring area. Consequences of this with regard to genetic variation along the transect are briefly discussed.     

Steroid transformation by activated living immobilized Arthrobacter simplex cells

A preparation of living Arthrobacter simplex cells immobilized in polyacrylamide gel, which showed steroid-Δ¹-dehydrogenase activity, was studied. The entrapped microorganisms catalyzed the transformation of cortisol to prednisolone and this reaction was followed spectrophotometrically or with the aid of thin layer chromatography (TLC) and high pressure liquid chromatography (HPLC). About 40% of the original activity found with free bacteria was retained after immobilization. The steroid dehydrogenase activity of polyacrylamide-entrapped A. simplex could be raised to a minor extent in alcoholic solvents or by addition of a cofactor such as menadione. On incubation in various nutrient media, on the other hand, the activity could be increased considerablyl, usually 7–10 times. Possible causes for the observed increase in activity have been investigated, and microbial growth of the original entrapped microorganisms appears to be the major reason. Frozen activated preparations of immobilized A. simplex showed only a small loss of activity on storage for at least four months. A semicontinuous batch wise operation with immobilized A. simplex in different nutrient media was carried out. At the end of the experiment the steroid transformation capacity was 0.5 g steroid per day per g gel (wet weight).     

Bead-to-bead transfer of chinese hamster ovary cells using macroporous microcarriers

Chinese hamster ovary cells (CHO-K1) were cultivated in macroporous gelatin microcarriers (CultiSpher G and CultiSpher S) in spinner flasks and a 5 1 bioreactor. Near-to-confluent cultures were harvested by bead-to-bead transfer where intact microcarriers with cells were transferred from a spinner flask to another spinner flask or to the bioreactor with naked microcarrier beads. Successful bead-to-bead transfer was achieved in various split ratios. The duration of attachment seemed to be important where the direct contact of beads to each other can be achieved by intermittent stirring. Repeated transfers were performed and at least four transfers in spinner flasks were achieved.Two variations of bead-to-bead transfer were performed in the 5 1 bioreactor either by seeding the bioreactor with near-to-confluent beads cultivated in spinner flasks orin situ transfer by adding fresh beads to the bioreactor. As in the spinner case, attachment was achieved by intermittent stirring where donor beads were in close proximity to the acceptor beads. Again successful transfers were obtained as evidenced by the good growth on acceptor beads where cell yields were in the range of 3100–4500 cells/bead.The results suggest that bead-to-bead transfer of CHO-K1 cells can be easily performed and do provide an alternative route to applications where dissolution techniques may not offer an efficient solution.