Carbohydrate-dependent binding of the cell-free hemagglutinin of Vibrio cholerae to glycoprotein and glycolipid.

The carbohydrate-binding specificity of the cell-free hemagglutinin (HA) of Vibrio cholerae (K.K. Banerjee, A.N. Ghose, K. Datta-Roy, S.C. Pal, and A.C. Ghose, Infect. Immun.58:3698-3705, 1990) was studied by using glycoconjugates with defined sugar sequences. The HA was not inhibited by simple sugars including glucobiose, galabiose, and their N-acetylated derivatives. The hemagglutination of rabbit erythrocytes by the HA was inhibited moderately by fetuin, calf thyroglobulin, and human alpha 1-acid glycoprotein, all of which contain multiple asparagine-linked complex-type oligosaccharide units alone or in combination with serine/threonine-linked oligosaccharide units. The inhibitory potencies of the glycoproteins increased approximately 10-fold following removal of the terminal sialic acid and were completely destroyed by exhausative proteolysis. The HA agglutinated phosphatidylcholine liposomes containing GM1-ganglioside or its asialo-derivative in the presence of Ca2+ ions. The association constants of the complexes of the HA with asialofetuin, asialothyroglobulin, GM1-ganglioside, and asialo-GM1-ganglioside were determined by an enzyme-linked immunosorbent assay-based assay and found to be 1.7 x 10(7) M-1, 1.5 x 10(7) M-1, 1.8 x 10(7) M-1, and 2.4 x 10(7) M-1, respectively. Studies using chemically modified glycoproteins and plant lectins with defined sugar specificity revealed that the HA recognized the terminal beta 1-galactosyl moiety of these glycoconjugates. There was no evidence for the presence of an extended carbohydrate-binding domain in the HA molecule or a preference of the HA for a complex, branched oligosaccharide structure. Similar to the mechanisms proposed for the binding of cholera toxin and Shiga toxin to glycolipids and neoglycoproteins, the strong interaction of V. cholerae cell-free HA with glycoconjugates appeared to be a consequence of multiple weak binding to terminal beta1-galactosyl moieties of the glycoproteins or glycolipids.     

Method for enhancing solubility of the expressed recombinant proteins in Escherichia coli

The production of correctly folded protein in Escherichia coli is often challenging because of aggregation of the overexpressed protein into inclusion bodies. Although a number of general and protein-specific techniques are available, their effectiveness varies widely. We report a novel method for enhancing the solubility of overexpressed proteins. Presence of a dipeptide, glycylglycine, in the range of 100 mM to 1 M in the medium was found to significantly enhance the solubility (up to 170-fold) of the expressed proteins. The method has been validated using mycobacterial proteins, resulting in improved solubilization, which were otherwise difficult to express as soluble proteins in E. coli. This method can also be used to enhance the solubility of other heterologous recombinant proteins expressed in a bacterial system.     

Effect of heart rate on hemodynamic endpoints under concomitant microvascular disease in a porcine model

Diagnosis of the ischemic power of epicardial stenosis with concomitant microvascular disease (MVD) is challenging during coronary interventions, especially under variable hemodynamic factors like heart rate (HR). The goal of this study is to assess the influence of variable HR and percent area stenosis (%AS) in the presence of MVD on pressure drop coefficient (CDP; ratio of transstenotic pressure drop to the distal dynamic pressure) and lesion flow coefficient (LFC; ratio of %AS to the CDP at the throat region). We hypothesize that CDP and LFC are independent of HR. %AS and MVD were created using angioplasty balloons and 90-μm microspheres, respectively. Simultaneous measurements of pressure drop (DP) and velocity were done in 11 Yorkshire pigs. Fractional flow reserve (FFR), CDP, and LFC were calculated for the groups HR < 120 and HR > 120 beats/min, %AS < 50 and %AS > 50, and additionally for DP < 14 and DP > 14 mmHg, and analyzed using regression and ANOVA analysis. Regression analysis showed independence between HR and the FFR, CDP, and LFC while it showed dependence between %AS and the FFR, CDP, and LFC. In the ANOVA analysis, for the HR < 120 beats/min and HR > 120 beats/min groups, the values of FFR (0.82 ± 0.02 and 0.82 ± 0.02), CDP (83.15 ± 26.19 and 98.62 ± 26.04), and LFC (0.16 ± 0.03 and 0.15 ± 0.03) were not significantly different (P > 0.05). However, for %AS < 50 and %AS > 50, the FFR (0.89 ± 0.02 and 0.75 ± 0.02), CDP (35.97 ± 25.79.10 and 143.80 ± 25.41), and LFC (0.09 ± 0.03 and 0.22 ± 0.03) were significantly different (P < 0.05). A similar trend was observed between the DP groups. Under MVD conditions, FFR, CDP, and LFC were not significantly influenced by changes in HR, while they can significantly distinguish %AS and DP groups.     

Polyomavirus Large T Antigen Binds Symmetrical Repeats at the Viral Origin in an Asymmetrical Manner

Polyomaviruses have repeating sequences at their origins of replication that bind the origin-binding domain of virus-encoded large T antigen. In murine polyomavirus, the central region of the origin contains four copies (P1 to P4) of the sequence G(A/G)GGC. They are arranged as a pair of inverted repeats with a 2-bp overlap between the repeats at the center. In contrast to simian virus 40 (SV40), where the repeats are nonoverlapping and all four repeats can be simultaneously occupied, the crystal structure of the four central murine polyomavirus sequence repeats in complex with the polyomavirus origin-binding domain reveals that only three of the four repeats (P1, P2, and P4) are occupied. Isothermal titration calorimetry confirms that the stoichiometry is the same in solution as in the crystal structure. Consistent with these results, mutation of the third repeat has little effect on DNA replication in vivo. Thus, the apparent 2-fold symmetry within the DNA repeats is not carried over to the protein-DNA complex. Flanking sequences, such as the AT-rich region, are known to be important for DNA replication. When the orientation of the central region was reversed with respect to these flanking regions, the origin was still able to replicate and the P3 sequence (now located at the P2 position with respect to the flanking regions) was again dispensable. This highlights the critical importance of the precise sequence of the region containing the pentamers in replication.     

Localization and origin of the intestinal peroxidase--effect of adrenal glucocorticoids

Peroxidase activity in rat intestine is stimulated two-fold after bilateral adrenalectomy and is reversed by dexamethasone (9-fluoro-11 beta,17,21-trihydroxy-16 alpha-methyl-1-4-pregnadiene-3,20-dione). The enzyme activity is inhibited on administration of various glucocorticoids of which dexamethasone acts as the most potent inhibitor of the enzyme in vivo. The change of enzyme activity results neither from alteration of the apparent Km of the enzyme nor from enzyme synthesis. Although a small amount of peroxidase is located in the intestinal epithelial cells, a large amount is present in the rest of the intestine. Histochemical studies indicate the presence of peroxidase in the lamina propria, the core of the intestinal villi which contains eosinophil. The peroxidase isolated from the epithelial cell-free intestine is similar to the peroxidase obtained from the pure eosinophil in terms of various physicochemical properties. Dexamethasone also inhibits the eosinophil peroxidase and decreases the number of both circulating and intestinal eosinophil. Studies indicate that a large part of the peroxidase of the intestine is contributed by invading eosinophil and dexamethasone inhibits the enzyme by sequestration of eosinophil both from intestine and blood possibly to the peripheral lymph nodes.     

Bandwidth Enhancement of Planar Terahertz Metasurfaces via Overlapping of Dipolar Modes

Plasmonics - We present enhancement of operational bandwidths of planar terahertz metasurfaces by incorporating a complex unit cell that consists of a pair of concentric ring resonators. The inner...