We show how the water content (and effective density) of thin adsorbed films composed of biomolecules can be determined using combined quartz crystal microbalance with dissipation monitoring (QCM-D) and surface plasmon resonance (SPR) analysis. In particular, these techniques, combined with theoretical treatment using a Voigt-based viscoelastic model, were applied to analyze the state of surface immobilized single stranded biotin-modified probe DNA (b-DNA) coupled via streptavidin to a biotin-doped supported phospholipid bilayer (b-SPB)). From a proper analysis, it is demonstrated how changes in effective thickness, delta(f), and the viscoelastic components (shear viscosity, eta(f), and shear elasticity, mu(f))) can be obtained during both DNA immobilization and hybridization with single stranded fully complementary target DNA. In particular, it is demonstrated how this type of analysis can be used to control the state of streptavidin arrangement for improved measurements of DNA hybridization kinetics. The latter is demonstrated by identifying a surface-coverage dependent viscoelastic behavior of immobilized b-DNA, which is shown to influence the hybridization efficiency.

Simultaneous quartz crystal microbalance with dissipation monitoring (QCM-D) and surface plasmon resonance (SPR) measurements are used to analyze the surface kinetics of two biomacromolecular systems, one lipid and one protein based, undergoing surface-induced conformational changes. First we establish a theoretical platform, which allows quantitative analysis of the combined SPR and QCM-D data. With this theoretical base, new information can be extracted, not obtainable with either technique alone. As an example we demonstrate how time-resolved measurements with these two techniques in combination--yielding three independent measured quantities--add new information about (i) kinetics, i.e. number of adsorbed molecules per unit area versus time, and (ii) temporal variation in the mass fraction of coupled water versus coverage. In particular, it is demonstrated for the first time, how the kinetics of the process during which adsorbed vesicles are spontaneously transformed into a supported phospholipid bilayer (SPB) on SiO(2) can be quantitatively separated into its two dominating states: adsorbed vesicles and supported planar bilayer patches. In addition, the relevance of dynamically coupled water for interpretation and modeling of the QCM-D response during bilayer formation is discussed and further illustrated with a second model system: streptavidin adsorption on a biotin-modified SPB. A very strong coverage dependence in the number of water molecules per protein sensed by the QCM is demonstrated, with strong implications for the use of QCM as a tool for quantitative determination of protein mass uptake kinetics.

Quartz crystal microbalance with dissipation (QCM-D) measurements were used to investigate the adsorption of human fibrinogen, human serum albumin, bovine hemoglobin, horse heart cytochrome c, human immunoglobulin (hIgG), and 10% fetal bovine serum on supported bilayers of egg-phosphatidylcholine (eggPC) lipids. For comparison the adsorption of fibrinogen and hIgG to eggPC bilayers was also studied with surface plasmon resonance (SPR). The supported bilayers were formed in situ by vesicle adhesion and spontaneous fusion onto a SiO 2 surface. The supported lipid bilayer is highly protein resistant: The irreversible adsorption measured with the QCM-D technique was below the detection level, while reversible protein adsorption was detected for all the proteins in the range 0.3-4% of the saturation coverage on a hydrophobic thiol monolayer on gold. The adsorbed amounts were slightly higher for the SPR measurements. Possible mechanisms for the protein resistance of eggPC bilayers are briefly discussed. C 2002 Elsevier Science

SummaryThe R28 protein is a surface molecule expressed by some strains of Streptococcus pyogenes (group A streptococcus). Here, we present evidence that R28 may play an important role in virulence. Sequence analysis demonstrated that R28 has an extremely repetitive sequence and can be viewed as a chimera derived from the three surface proteins Rib, a and b of the group B streptococcus (GBS). Thus, the gene encoding R28 may have originated in GBS. The R28 protein promotes adhesion to human epithelial cells, as shown by experiments with an R28-negative mutant and by the demonstration that antibodies to highly puri®ed R28 inhibited adhesion. In a mouse model of lethal intraperitoneal S. pyogenes infection, antibodies to R28 conferred protective immunity. However, the virulence of an R28-negative mutant was similar to that of the parental strain in the intraperitoneal infection model. Together, these data indicate that R28 represents a novel type of adhesin expressed by S. pyogenes and that R28 may also act as a target for protective antibodies at later stages of an infection. We consider the hypothesis that R28 played a pathogenetic role in the well-known epidemics of childbed fever (puerperal fever), which were caused by S. pyogenes. A role for R28 in these epidemics is suggested by epidemiological data.

Much effort is currently concentrated on research devoted to biofunctional patterned surfaces, which constitute the fundament for the development of microarrays for high-throughput gene and protein analyses. DNA microarrays have proved very successful, [1] and the concept is in the process of being applied to protein arrays. [2] However, in contrast to DNA fragments, proteins are easily denatured in contact with solid supports, and robotic printing of proteins onto chemically reactive glass slides [3] will not necessarily be applicable as a generic protocol for the preparation of protein arrays. Supported phosphatidylcholine lipid bilayers have emerged as interesting candidate substrates for protein chips, since they efficiently reduce nonspecific protein adsorption [4, 5] and, at the same time, allow different strategies for protein immobilization with biospecific water, desalted with a NAP5 column (Amersham Pharmacia, USA) according to manufacturer's protocols, and stored as working stock solutions at À 20 8C until use. Epoxy-derivatized slides were prepared from plain glass slides (Sigma, USA) as previously described. [9] Nhydroxysuccinimide slides were also used to spot the proteins but consistently gave inferior results. Proteins were prepared in NaHCO 3 buffer (0.1 M, pH 9) and arrayed on epoxy slides with a spacing of 180 mm between the spots by using an statistical microarray analysis arrayer (Engineering Services Inc., Ontario, Canada). After a 2-hour incubation period the slides were either used immediately, or stored for future use at 4 8C. The slides, if stored, were typically used within 48 h of printing.Unless otherwise indicated, probing and reactions on slides were performed as follows: Before use, the slides were quenched by treatment with phosphate-buffered saline (PBS) and glycine (0.5 M) on a shaker for 10 min. The slides were blocked with PBS, glycine (0.5 M), and bovine serum albumin (BSA; 1 % w/v) for 20 min, then washed with distilled water and air dried. The labeled probe was then applied: a mixture containing the probe (2 mM) was prepared by adding stock probe solution (0.5 mL, 200 mM) to tris(hydroxymethyl)aminomethane (Tris) buffer (49 mL, 50 mM, pH 8), and BSA (0.5 mL, 1 % w/v). The resulting mixture was applied to each slide by the coverslip method [9] and incubated for 30 min in the dark. The excess probe was washed off after incubation with distilled water, and the slides were subsequently washed with PBS that contained Tween (0.2 % v/v) for 15 minutes on a shaker. The slides were then washed with distilled water, air dried, and scanned with an ArrayWorx microarray scanner (Applied Precision, USA) at 548/595 nm. For the PMSF experiment, each slide was first incubated with freshly prepared PMSF (50 mL, 1 mM in 50 mM Tris, pH 8) for 30 minutes, rinsed extensively with distilled water to remove any free residual PMSF, and screened with FP-Cy3. The inhibition experiments were identical to the probe ± enzyme reactions, except that varying concentrations of trypsin inhibitor (original...

Mosaicism seems to mitigate the TS phenotype and the cardiovascular risk factor profile. Mosaics were diagnosed 8 years later than 45,X cases. This emphasizes the necessity for a stricter genotype categorization not only in the clinic but also in research on TS than previously adopted.

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The standard procedure of estimation of obstetric bleeding was found to be unreliable. In this study, blood loss was over-estimated in cesareans. In vaginal deliveries, there seemed to be no correlation. Estimated blood loss as a quality indicator or as a variable in studies comparing complications must be used with caution. For clinical purposes, estimation of blood loss and measurement of post partum hemoglobin is of low value and may lead to the wrong conclusions.