Unfolding of acrylodan-labeled human serum albumin probed by steady-state and time-resolved fluorescence methods

Steady-state and time-resolved fluorescence spectroscopy was used to follow the local and global changes in structure and dynamics during chemical and thermal denaturation of unlabeled human serum albumin (HSA) and HSA with an acrylodan moiety bound to Cys34. Acrylodan fluorescence was monitored to obtain information about unfolding processes in domain I, and the emission of the Trp residue at position 214 was used to examine domain II. In addition, Trp-to-acrylodan resonance energy transfer was examined to probe interdomain spatial relationships during unfolding. Increasing the temperature to less than 50 degrees C or adding less than 1.0 M GdHCl resulted in an initial, reversible separation of domains I and II. Denaturation by heating to 70 degrees C or by adding 2.0 M GdHCl resulted in irreversible unfolding of domain II. Further denaturation of HSA by either method resulted in irreversible unfolding of domain I. These results clearly demonstrate that HSA unfolds by a pathway involving at least three distinct steps. The low detection limits and high information content of dual probe fluorescence should allow this technique to be used to study the unfolding behavior of entrapped or immobilized HSA.     

Discerning intra‐tuber differences in textural properties in cooked Solanum tuberosum group Tuberosum and group Phureja tubers

BACKGROUND: The textural properties of potato tubers influence their acceptability and palatability and these properties differ between varieties, groups and progeny. The aim of this study was to compare the textural properties of cooked tubers of Solanum tuberosum group Phureja with those of group Tuberosum. RESULTS: To assess intra‐tuber differences, the textural properties of seven cubes from defined positions along the longitudinal axis of tubers of four Tuberosum group cultivars and three Phureja group lines were tested after cooking using an amended wedge fracture method. Tuberosum group tubers gave consistently higher peak force and work done values during fracture than the Phureja group tubers. Moreover, the values for cubes 1–6 from any tuber were not significantly different and only cube 7, from the stem end, gave higher values. Therefore, the use of any of cubes 1–6 is a valid measurement of the tuber as a whole but the central cube 4 may be most conveniently located. The dry matter content of the cubes did not influence the textural properties of the cubes, which suggested that starch swelling is not the main driving force for textural differences. Total pectin methyl esterase (PME) activity was consistently higher in cubes of the Tuberosum group cultivars over the Phureja group lines. CONCLUSION: The method developed is valid and consistent for assessing textural differences within potato germplasm. The relationship between PME activity and enhanced resistance to fracture suggests that PME may modulate pectin cohesiveness, perhaps through increasing Ca²⁺‐bridges, to provide greater resistance to fracture. Copyright © 2010 Society of Chemical Industry     

3,4,3',4'-tetrachloroazobenzene: a potential environmental toxicant

3,4,3',4'-tetrachloroazobenzene (TCAB), a potential environmental toxicant formed in the industrial manufacturing and the microbial degradation of chloroanilide herbicides, has been prepared in 87% yield by an improved procedure. In vitro experiments with C3H/10T1/2 cells have shown that TCAB is toxic to mammalian cells, which may be correlated with observable cytological changes. TCAB has also been found to be weakly mutagenic by the Salmonella/microsome test. In addition, C3H/10T1/2 cells are transformed following a ten day incubation with TCAB. These data suggest the potential health hazard associated with the environmental occurrence of TCAB.     

Accessibility of the fluorescent reporter group in native, silica-adsorbed, and covalently attached acrylodan-labeled serum albumins

Fluorescence quenching techniques are used to investigate the accessibility of a model biorecognition element-reporter group system when in buffer, surface-adsorbed, and covalently attached to a silica surface. The site-selective fluorescent reporter group, 6-acryloyl(dimethylamino)naphthalene (acrylodan, Ac), is attached covalently (at cysteine-34) to bovine and human serum albumin (BSA and HSA, respectively) and serves as a surrogate recognition element-reporter group system. Molecular oxygen is used to quench the Ac fluorescence and the accessibility, in the form of bimolecular rate constants (kq), in each model system is quantified. Although one might expect these systems to exhibit similar behavior, differences in quenching characteristics are observed, such as wavelength dependency of the Stern-Volmer quenching constant (KSV) for the native proteins in buffer. BSA-Ac exhibits wavelength dependent KSV values as well as a blue-shifted emission spectrum on O2 addition. Physisorption of BSA-Ac onto a fused-silica optical fiber lowers the accessibility of Ac to O2, whereas covalent attachment of BSA-Ac to APTES/glutaraldehyde-modified silica enhances the accessibility of the Ac reporter group to O2.     

Pathogenetic effector function of CD4-positive T helper 1 cells in hepatitis B virus transgenic mice

The pathogenetic effector functions of hepatitis B virus (HBV)-specific CD4+, Th1 cells were analyzed in two inbred lineages of HBV transgenic mice, one of which overexpresses the HBV large envelope protein rendering the hepatocytes hypersensitive to the cytopathic effects of IFN-gamma, and another that expresses all of the HBV proteins and replicates the virus in the liver. Transfer of HBV envelope-specific Th1 cells resulted in recognition of viral Ag expressed by hepatic nonparenchymal cells, cytokine release, and a transient necroinflammatory liver disease in both lineages. The liver disease was very severe in the IFN-gamma-sensitive lineage, and it was less severe in the lineage that replicates the HBV genome; nonetheless, in this lineage the Th1 cytokines produced by these cells suppressed viral replication in the liver. These results demonstrate that CD4+ T cells with a Th1 functional phenotype can perform pathogenetic and antiviral effector functions in vivo. This suggests that CD4+ T cells can contribute directly to disease pathogenesis and inhibit viral replication during HBV infection.     

Case report: bladder opacification following enteral iopamidol as a sign of occult intestinal infarction

A case of intestinal infarction due to superior mesenteric artery embolism is described in a 30-year-old female with Crohn's disease. This was accompanied by bladder opacification following enteral iopamidol administered at endoscopic retrograde cholangiopancreatography. The likely mechanism of this finding is transmural and transperitoneal absorption of enteral contrast medium and its significance as a sign of mesenteric ischaemia is discussed.