One- and two-photon induced fluorescence of Pacific Blue-labeled human serum albumin deposited on different core size silver colloids

We studied one- and two-photon induced fluorescence of Pacific Blue (PB)-labeled human serum albumin (HSA) in the presence of different size silver colloids. The PB fluorescence emission intensity was observed with small (30-40 nm) and large (about 120 nm) colloids and compared with PB emission in absence of colloids. For the system with a small core size colloids we did not detect any fluorescence enhancement with one-photon excitation and the enhancement observed with two-photon excitation was about 2.5-fold. In contrast, for large silver colloids we observed about a 2-fold increase in PB fluorescence brightness for one-photon excitation, and the enhancement with two-photon excitation excided 13-folds. Much stronger increases in brightness observed with two-photon excitation, compared to one-photon excitation, indicate a dominant role of enhanced local field in fluorescence enhancement on silver colloids in solutions.     

Phosphorylation-dependent phospholipase D activity of matrix vesicles

A progressive hydrolysis of phospholipids was observed during the mineralization process mediated by extracellular matrix vesicles. Increasing levels of different hydrolysis products revealed phospholipase A and D activities. The importance of these enzymes for the mineralization process lies in a high rate of hydrolysis of neutral phospholipids and lower rate of degradation of anionic phospholipids, which may favor mineral formation in vesicular membrane and membrane breakdown necessary for the release of mineral deposits into extracellular matrix. In this report, we focus on the phosphorylation-dependent phospholipase D activity during mineral formation initiated by chicken embryo matrix vesicles.     

Decrease of serum chemerin concentration in patients with end stage renal disease after successful kidney transplantation

Chemerin is an adipokine associated with metabolic syndrome, systemic inflammation and innate immune system. It has been suggested recently that the decrease in renal function may cause an increase in serum chemerin concentration. In this paper we investigated the effect of kidney transplantation on elevated serum chemerin concentration in dialyzed patients with end stage renal disease (ESRD). Twenty five ESRD patients were tested before and 3months after the kidney transplantation. The control group was comprised of twenty one healthy subjects. Serum chemerin concentrations were measured using commercial ELISA kit, and were related to clinical status, and biomarkers of renal function. We have shown that the kidney transplantation resulted in the decrease of the serum chemerin concentration. Concomitantly, serum creatinine, blood urea nitrogen, phosphate and C-reactive protein concentrations were significantly reduced, while estimated glomerular filtration rate (eGFR), calcium and hemoglobin substantially increased. Univariate regression analysis showed that serum chemerin concentration was positively correlated with serum creatinine and phosphate concentrations and negatively correlated with eGFR. The results presented here indicate that the serum chemerin concentration in patients with ESRD normalizes after the kidney transplantation, and provide additional evidence that serum chemerin concentration is related to renal function.     

Identification of the first prokaryotic collagen sequence motif that mediates binding to human collagen receptors, integrins alpha2beta1 and alpha11beta1

Many pathogenic bacteria interact with human integrins to enter host cells and to augment host colonization. Group A Streptococcus (GAS) employs molecular mimicry by direct interactions between the cell surface streptococcal collagen-like protein-1 (Scl1) and the human collagen receptor, integrin alpha2beta1. The collagen-like (CL) region of the Scl1 protein mediates integrin-binding, although, the integrin binding motif was not defined. Here, we used molecular cloning and site-directed mutagenesis to identify the GLPGER sequence as the alpha2beta1 and the alpha11beta1 binding motif. Electron microscopy experiments mapped binding sites of the recombinant alpha2-integrin-inserted domain to the GLPGER motif of the recombinant Scl (rScl) protein. rScl proteins and a synthetic peptide harboring the GLPGER motif mediated the attachment of C2C12-alpha2+myoblasts expressing the alpha2beta1 integrin as the sole collagen receptor. The C2C12-alpha11+myoblasts expressing the alpha11beta1 integrin also attached to GLPGER-harboring rScl proteins. Furthermore, the C2C12-alpha11+cells attached to rScl1 more efficiently than C2C12-alpha2+cells, suggesting that the alpha11beta1 integrin may have a higher binding affinity for the GLPGER sequence. Human endothelial cells and dermal fibroblasts adhered to rScl proteins, indicating that multiple cell types may recognize and bind the Scl proteins via their collagen receptors. This work is a stepping stone toward defining the utilization of collagen receptors by microbial collagen-like proteins that are expressed by pathogenic bacteria.     

The Scl1 protein of M6-type group A Streptococcus binds the human complement regulatory protein, factor H, and inhibits the alternative pathway of complement

Non-specific activation of the complement system is regulated by the plasma glycoprotein factor H (FH). Bacteria can avoid complement-mediated opsonization and phagocytosis through acquiring FH to the cell surface. Here, we characterize an interaction between the streptococcal collagen-like protein Scl1.6 of M6-type group A Streptococcus (GAS) and FH. Using affinity chromatography with immobilized recombinant Scl1.6 protein, we co-eluted human plasma proteins with molecular weight of 155 kDa, 43 kDa and 38 kDa. Mass spectrometry identified the 155 kDa band as FH and two other bands as isoforms of the FH-related protein-1. The identities of all three bands were confirmed by Western immunoblotting with specific antibodies. Structure-function relation studies determined that the globular domain of the Scl1.6 variant specifically binds FH while fused to collagenous tails of various lengths. This binding is not restricted to Scl1.6 as the phylogenetically linked Scl1.55 variant also binds FH. Functional analyses demonstrated the cofactor activity of the rScl1.6-bound FH for factor I-mediated cleavage of C3b. Finally, purified FH bound to the Scl1.6 protein present in the cell wall material obtained from M6-type GAS. In conclusion, we have identified a functional interaction between Scl1 and plasma FH, which may contribute to GAS evasion of complement-mediated opsonization and phagocytosis.     

Conformational studies of hexapeptides containing two dehydroamino acid residues in positions 2 and 5 in peptide chain

Conformational preferences of a group of hexapeptides containing two dehydroamino acid residues in Positions 2 and 5 in peptide chain were investigated by means of spectroscopic methods (NMR and CD) and theoretical calculations. In the case of dimethylsulfoxide (DMSO) solution, only peptide with free N-termini adopted rigid 3(10)-helical conformation, for the rest of examined peptides extended and "zig-zag" conformers were predominant. CD measurements showed that only in chloroform solution the conformational freedom of investigated peptides was restricted.     

Lipids and fatty acids in the copepod Jaschnovia brevis (Jaschnov) and in particulates from Arctic waters

The small, sub-ice copepod Jaschnovia brevis is rich in triacylglycerols, suggesting a feeding behaviour not constrained to the seasonal phytoplankton bloom. The copepod's triacylglycerol reserves contain: the diatom biomarkers 16:1n-7 (23.9%), 20:5n-3 (8.5%) and C16 PUFA (1.3%), the flagellate biomarkers 18:4n-3 (3.7%) and 22:6n-3 (3.3%), and the Calanus copepod biomarkers 20:1n-9 (7.7%) and 22:1n-11 (6.2%). Total lipid from particulates in the water column contained polar lipid (45.0%), wax esters (24.9%) and triacylglycerols (11.2%) as major components. The total lipids in the particulates were rich in 18:1n-9 (31.5%) and 16:0 (21.2%), and relatively rich in 18:0 (7.8%) and 18:2n-6 (9.2%). The triacylglycerols in the particulates contained 16:1n-7 (20.7%), C16 PUFA (4.1%), 18:4n-3 (1.9%), 20:5n-3 (3.6%), 22:6n-3 (1.9%), 20:1n-9 (5.2%) and 22:1n-11 (3.9%). The polar lipids in the particulates contained 16:1n-7 (17.3%), C16 PUFA (7.8%), 18:4n-3 (3.3%), 20:5n-3 (14.5%) and 22:6n-3 (9.6%). The fatty alcohols in the wax esters of the particulates were mainly 16:0 (11.3%), 20:1n-9 (21.1%) and 22:1n-11 (30.6%). The nature of the particulates, their possible origin in living and non-living material, and their role in the nutrition of J. brevis are discussed.     

Fluorescence and Phosphorescence Study of Tet Repressor–Operator Interaction

Fluorescence and phosphorescence measurements have been carried out on single-p tryptophan (Trp 43 or Trp 75)-containing mutants of Tet repressor (Tet R). Tet R containing Trp 43, the residue localized in the DNA recognition helix of the repressor, has been used to observe the binding of Tet R to two 20-bp DNA sequences of tet O₁ and tet O₂ operators. Binding of Tet R to tet O₁ operator leads to a 78% decrease of the repressor fluorescence intensity, with an accompanying 20-nm blue shift of its fluorescence emission maximum to 330 nm. Upon binding of Tet R to tet O₂ operator, the Trp 43 fluorescence intensity is quenched by 60%, and a 10-nm shift of its emission maximum to 340 nm occurs. Solute fluorescence quenching studies, using acrylamide, performed at low ionic strength indicate that in both the complex of Tet R with the O₁ and that with the O₂ operator, Trp 43 is moderately buried, as indicated by a bimolecular rate quenching constant of about 1.8 × 10⁹ M^–1 sec^–1. In contrast to the Tet R–tet O₂ complex, the Stern–Volmer acrylamide quenching constant K _sv of the complex with tet O₁ operator changes from 7.5 M^–1 at 5 mM NaCl to 22 M^–1 at 200 mM NaCl, indicating different exposures of Trp 43 in the two complexes in solutions of higher ionic strength. Phosphorescence studies showed a 0–0 vibronic transition at 408 and 403 nm for Trp 43 and Trp 75, respectively. Upon binding of Tet R to the tet operators, we observed red shifts of 0–0 vibronic bands of Trp 43 to 413 and 412 nm for tet O₁ and tet O₂ operator, respectively, and the phosphorescence triplet lifetime of Trp 43 at 75 K was quenched from 6.0–5.5 to 3.5–3.3 sec. The thermal phosphorescence quenching profile ranged from –200°C to –20°C, and differed drastically for the two complexes, suggesting different dynamics of the microenvironment of the Trp 43 residue. The luminescence data for Trp 43 of Tet R suggest that the recognition helix of Tet R interacts in different fashions with the tet O₁ and tet O₂ operators.