Electrochemistry and electrocatalysis with heme proteins in chitosan biopolymer films

Protein-chitosan (CS) films were made by casting a solution of proteins and CS on pyrolytic graphite electrodes. Myoglobin (Mb), hemoglobin (Hb), and horseradish peroxidase (HRP) incorporated in CS films gave a pair of stable, well-defined, and quasi-reversible cyclic voltammetric peaks at about -0.33V vs saturated calomel electrode in pH 7 buffers, respectively, while catalase (Ct) in CS films showed a peak pair at about -0.46V which was not stable. All these peaks are located at the potentials characteristic of heme Fe(III)/Fe(II) redox couples of the proteins. The electrochemical parameters such as formal potentials (E degrees (')) and apparent heterogeneous electron-transfer rate constants (k(s)) were estimated by square-wave voltammetry with nonlinear regression analysis. Chitosan films contained considerable water and formed hydrogel in aqueous solution. Positions of the Soret absorbance band suggest that Mb and Hb in CS films keep their secondary structure similar to the native states in the medium pH range, while HRP and Ct retain their native conformation at least in the dry CS films. Scanning electron microscopy of the films demonstrated that interaction between the proteins and CS would make the morphology of dry protein-CS films very different from the CS films alone. Oxygen, trichloroacetic acid, nitrite, and hydrogen peroxide were catalytically reduced by all four proteins in CS films.     

Mitochondrial gene rearrangements confirm the parallel evolution of the crab-like form

The repeated appearance of strikingly similar crab-like forms in independent decapod crustacean lineages represents a remarkable case of parallel evolution. Uncertainty surrounding the phylogenetic relationships among crab-like lineages has hampered evolutionary studies. As is often the case, aligned DNA sequences by themselves were unable to fully resolve these relationships. Four nested mitochondrial gene rearrangements--including one of the few reported movements of an arthropod protein-coding gene--are congruent with the DNA phylogeny and help to resolve a crucial node. A phylogenetic analysis of DNA sequences, and gene rearrangements, supported five independent origins of the crab-like form, and suggests that the evolution of the crab-like form may be irreversible. This result supports the utility of mitochondrial gene rearrangements in phylogenetic reconstruction.     

The HIV-1 vaccine race

There is an urgent need to come up with a vaccine that will curtail the spread of HIV-1. To be successful, the scientific community must work in concert to develop sound strategies based on a deeper understanding of the virus. Significant technological advances are also required to overcome the unique obstacles posed by HIV-1. Some of the challenges we face in this endeavor are discussed here.     

ApoE-dependent sterol efflux from macrophages is modulated by scavenger receptor class B type I expression

Macrophages express a number of proteins involved in sterol efflux pathways, including apolipoprotein E (apoE) and scavenger receptor class B type I (SR-BI). We have investigated a potential interaction between these two sterol efflux pathways in modulating overall macrophage sterol flux. We utilized an experimental system in which we increased expression of each of these proteins to a high physiologic range in order to perform our evaluation. We show that in apoE-expressing cells, a 4-fold increase in SR-BI expression leads to reduction of sterol and phospholipid efflux. SR-BI-mediated reduction in sterol efflux was only observed in cells that expressed endogenous apoE. In J774 cells that did not express apoE, a similar increase in SR-BI level led to increased sterol efflux. The divergent response of sterol efflux after increased SR-BI was maintained in the presence of a number of structurally diverse extracellular sterol acceptors. Increased SR-BI expression also enhanced sterol efflux to exogenously added apoE. Investigation of a potential mechanism for reduced efflux in apoE-expressing cells indicated that SR-BI expression reduced macrophage apoE by accelerating the degradation of newly synthesized apoE. This led to decreased secretion of apoE and reduced the fraction of apoE sequestered on the cell surface. Thus, enhanced SR-BI expression in macrophages can reduce the cellular level and secretion of apoE by accelerating degradation of the newly synthesized protein. This reduction of endogenous apoE is accompanied by reduced sterol efflux from macrophages.     

A novel allele in the promoter of the hepatic lipase is associated with increased concentration of HDL-C and decreased promoter activity

Hepatic lipase (HL) is a lipolytic enzyme involved in the metabolism of plasma lipoproteins, especially HDLs. Association of the polymorphisms in the promoter region of the LIPC gene to post-heparin plasma HL activity and the plasma HDL-C concentration has been investigated thoroughly, but to date little is known about this in the Chinese. In the present study, we analyzed the polymorphisms in the promoter region of LIPC gene in Chinese patients with coronary artery disease (CAD) using denaturing high performance liquid chromatography (DHPLC) and DNA sequencing. As the result, a novel single nucleotide polymorphism -586T-to-C was identified and no linkage of this variant with other polymorphisms in the promoter was found. Compared with the nonsymptomatic control subjects, excess of carriers of the -586T/C substitution were detected in the CAD patients (43% vs. 31%, chi(2) = 4.597, degree of freedom = 2, P = 0.032).The -586C allele carriers in the CAD patients had a significantly higher HDL-C level than the noncarriers (1.13 +/- 0.24 mmol/l vs. 0.91 +/- 0.14 mmol/l, P < 0.05). To test the functionality of this substitution, luciferase-reporter assays was performed in HepG2 cells. Promoter activity of the -586C construct was decreased 2-fold than the -586T construct. Our studies suggest that a T-to-C substitution at -586 of the LIPC promoter is associated with a lowered HL activity and that this variation may contribute to the increased plasma HDL-C concentration in the Chinese.     

Functional characterization of alternatively spliced 5-HT2 receptor isoforms from the pharynx and muscle of the parasitic nematode,Ascaris suum

Serotonin (5-HT) receptors play key regulatory roles in nematodes and alternatively spliced 5-HT2 receptor isoforms have been identified in the parasitic nematode, Ascaris suum. 5-HT2As1 and 5-HT2As2 contain different C-termini, and 5-HT2As1Delta4 lacks 42 amino acids at the C-terminus of the third intracellular loop. 5-HT2As1 and 5-HT2As2 exhibited identical pharmacological profiles when stably expressed in human embryonic kidney (HEK) 293 cells. Both 5-HT2As isoforms had higher affinity for 5-HT than their closely related Caenorhabditis elegans homolog (5-HT2Ce). This increased 5-HT affinity was not related to the substitution in 5-HT2As1 of F120 for Y in the highly conserved DRY motif found in the second intracellular loop of other 5-HT receptors, since a 5-HT2As1F120Y mutant actually exhibited increased 5-HT affinity compared with that of 5-HT2As1. As predicted, cells expressing either 5-HT2As1 or 5-HT2As2 exhibited a 5-HT-dependent increase in phosphatidylinositol (PI) turnover. In contrast, although 5-HT2As1Delta4 displayed a 10-fold higher affinity for 5-HT and 5-HT agonists than either 5-HT2As1 or 5-HT2As2, 5-HT2As1Delta4 did not couple to either PI turnover or adenyl cyclase activity. Based on RT-PCR, 5-HT2As1 and 5-HT2As2 were more highly expressed in pharynx and body wall muscle and 5-HT2As1Delta4 in nerve cord/hypodermis. This is the first report of different alternatively spliced 5-HT2 receptor isoforms from any system.