The analysis of heparin-protein interactions using evanescent wave biosensor with regioselectively desulfated heparins as the ligands

Evanescent wave biosensor has been recently employed as a powerful tool for analyses of macromolecular interactions. In the present study, evanescent wave biosensor analysis was developed to analyze the heparin-protein interaction using as ligands a series of heparin derivatives regioselectively desulfated by chemical methods, particularly to evaluate the effect of each sulfate group of heparin. The method for immobilizing heparin on the cuvette of the evanescent wave biosensor equipment was optimized to obtain the high response required for accurate measurement. The best result was achieved when the amino group introduced at the reducing end of heparin was coupled with carboxymethyl dextran on the surface of the cuvette using glycolchitosan as a multivalent linker. The established system appeared to describe well the interactions of heparin with such proteins as acidic and basic fibroblast growth factors and tissue factor pathway inhibitor.     

Comparison of photovoltaic behaviors for horseradish peroxidase and its mimicry by surface photovoltage spectroscopy

Surface photovoltage spectroscopy (SPS) was chosen to study the photovoltaic behavior of horseradish peroxidase (HRP), hemin and immobilized hemin (poly(NIPAAm/MBA/hemin)). Different photovoltaic behaviors were observed in these three systems. In air, similar SPS curves were found for HRP and poly(NIPAAm/MBA/hemin) with different response intensities. However, poly(NIPAAm/MBA/hemin) showed a wider changing range upon increasing the positive and negative bias to 1.0 V. The SPS of hemin showed a total different behavior when an external positive potential was applied. In vacuum, clearly different photovoltaic behaviors were found. Moreover, the response value decreased when HRP was exposed to O2, the SPS intensity was different from that in air, and could be altered by changing the external biases. On the other hand, the SPS could not be changed before and after poly(NIPAAm/MBA/hemin) was exposed to O2. These differences may result from different chemical microenvironments for hemin in HRP versus that in poly(NIPAAm/MBA/hemin). It could be concluded that H2O and O2 were important factors affecting the photovoltage response in HRP, but only H2O played this important role in poly(NIPAAm/MBA/hemin).     

Inhibition kinetics of green crab (Scylla serrata) alkaline phosphatase by zinc ions: a new type of complexing inhibition

The Tsou method was used to study the kinetic course of inactivation of green crab alkaline phosphatase by zinc ions. The results show that the enzyme was inactivated by a complexing scheme which has not been previously identified. The enzyme first reversibly and quickly binds Zn(2+) and then undergoes a slow reversible course to inactivation and slow conformational change. The inactivation reaction is a single molecule reaction and the apparent inactivation rate constant is for a saturated reaction being independent of Zn(2+) concentration if the concentration is sufficiently high. The microscopic rate constants of inactivation and the association constant were determined from the measurements.     

Uncoupling protein-2 participates in cellular defense against oxidative stress in clonal beta-cells

The role of uncoupling protein-2 (UCP-2) in beta-cells is presently unclear. We have tested the notion that UCP-2 participates in beta-cell defense against oxidants. Expression of the UCP-2 gene in clonal beta-cells (INS-1) was decreased by 45% after 48 h of culture with vitamin E and selenite. When INS-1 cells were exposed to 200 microM H(2)O(2) for 5 min, the cell viability (MTT assay) decreased to 85 +/- 1, 61 +/- 1, 40 +/- 2, and 39 +/- 2% of control when measured respectively 30 min, 2 h, 6 h, and 16 h after H(2)O(2) exposure. At corresponding time points UCP-2 mRNA levels were 1.01 +/- 0.09, 1.53 +/- 0.15 (P < 0.05), 1.44 +/- 0.18 (P = 0.06), and 1.12 +/- 0.09 fold of control, i.e., transiently increased. We next tested whether overexpression of UCP-2 could enhance resistance of beta-cells toward H(2)O(2) toxicity. A cotransfection method using EGFP as a suitable marker and a human cDNA UCP-2 construct was used for transient overexpression of UCP-2. Transfected cells expressed the gene about 30-fold more than normal cells. After exposure to H(2)O(2) (200 micrometer, 5 min), the survival of UCP-2 overexpressing cells was measured 30-45 min later by flow cytometry. Survival was 13 +/- 0.05% higher than control (EGFP only) cells, P < 0.004 for difference. The results indicate that oxidative stress induces UCP-2 expression in beta-cells, and that UCP-2 serves a role in beta-cell defense against oxidative stress.     

The rat H3 receptor: gene organization and multiple isoforms

Genomic DNA analysis revealed that the coding region of the rat histamine H3 receptor comprises three exons interrupted by two introns of approximately 1 kb each. Several H3 receptor mRNA variants were identified by PCR and cDNA cloning and sequencing. Four variants generated by pseudo-intron retention/deletion at the level of the third intracellular loop were designated H3(445), H3(413), H3(410), and H3(397), according to the length of their deduced amino acid sequence and display differential tissue expression. When expressed in CHO-K1 or Cos-1 cells, the H3(445), H3(413), and H3(397) were found to generate specific 125I iodoproxyfan binding of similar pharmacological profile. In addition, we identified two short variants, termed H3(nf1) and H3(nf2), which correspond to frame shift and stop codon interposition, respectively, and are presumably nonfunctional, among which H3(nf2) displays brain expression similar to that of the longer isoforms.     

Insulin-like growth factor-I promotes proliferation and migration of cavernous smooth muscle cells

To better understand the physiology of cavernous smooth muscle cells (CSMC), particularly their regulation by IGF-I, we isolated CSMC from rats of various ages and grew them as cell cultures. CSMC from very young (1 week of age) and very old (28 months of age) rats secreted the least amounts of IGF-I, and those from 16-week-old rats the most. IGF-I stimulated growth of CSMC at an optimal concentration of 12.5 ng/ml. At this concentration, CSMC from 11-week-old rats showed the highest growth rate and CSMC from 28-month-old rats showed the lowest. The optimal IGF-I concentration for migration of CSMC was 10 ng/ml. At this concentration, CSMC from 4-week-old rats showed the highest migratory rate and CSMC from 28-month-old rats showed the lowest. IGF-I also stimulated VEGF secretion from CSMC at an optimal concentration of 10 ng/ml. At this concentration, CSMC from 16-week-old rats secreted VEGF the most and CSMC from 28-month-old rats secreted the least. The expression levels of IGF-IR paralleled the IGF-I-regulated growth rates of these cells. Expression of IGF-IR was identified in the cavernous smooth muscle and the urethra epithelium of the penis.     

Differential regulation of interleukin-12 and interleukin-10 by heat shock response in murine peritoneal macrophages

Heat shock response is a conserved stress response and has been shown to have anti-inflammatory effects. We investigated the effect of heat shock response on LPS-induced production of IL-12 and IL-10, which are two important cytokines playing contradictory roles in regulation of immune response, by murine peritoneal macrophages. The data showed that induction of heat shock response strongly suppressed LPS-induced production of IL-12 while augmented that of IL-10, suggesting the pleiotropic effects of heat shock response on immune regulatory gene expression. Also, the novel observation on up-regulation of IL-10 by heat shock response adds to the mechanism by which heat shock response exerts its anti-inflammatory effects.     

Anion shielding of electrostatic repulsions in transthyretin modulates stability and amyloidosis: insight into the chaotrope unfolding dichotomy

The balance between stabilizing forces and the localized electrostatic repulsions destabilizing the transthyretin (TTR) tetramer is tunable via anion shielding. The two symmetrical anion interaction sites in TTR are comprised of residues Lys15 and Lys15' from opposing subunits on the periphery of the two thyroxine binding sites. These epsilon-ammonium groups repel one another and destabilize the tetramer, unless an appropriate anion is present, which stabilizes the tetramer. Chaotrope denaturation of TTR exhibits unusual behavior in that urea appears to be a stronger denaturant than GdmCl (guanidinium chloride), even though GdmCl is typically twice as powerful as a denaturant. The shift in the midpoint of the urea denaturation curve to higher concentrations as well as the increase in the mole fraction of tetramer that is highly resistant to denaturation with increasing KCl concentration provides strong evidence that anion shielding stabilizes the TTR tetramer. A consequence of tetramer stabilization is folding hysteresis, because the high GdmCl concentrations required to denature the anion-stabilized tetramer do not allow refolding of the unfolded monomers. The formation of amyloid fibrils by TTR requires that its normal tetrameric structure dissociate to alternatively folded monomers, a process mediated by acidification (pH 5-4). This process is inhibited by Cl(-) ions in a concentration-dependent fashion. Chloride ion may not be the relevant physiological TTR stability modulator, but it is the main focus of these studies explaining the hysteresis observed in the denaturation and refolding studies with GdmCl.