Leukotriene B4 receptor and the function of its helix 8

More than 30 lipid ligands, which express their biological activities through cognate G-protein-coupled receptors (GPCRs), have been reported. Among them, leukotriene B(4) (LTB(4)) is a potent lipid mediator involved in host defense, inflammation, and the immune responses. Two GPCRs for LTB(4) (BLT1 and BLT2) have been cloned and analyzed. Recent studies using genetically engineered mice suggest that BLT1 plays an important role in several inflammatory diseases including ischemic reperfusion tissue injury, atherosclerosis, and bronchial asthma. BLT1 is also a good tool to study the molecular mechanism of GPCR activation and inactivation in vitro. In this brief review, we focus on the biological and biochemical properties of BLT1 with special attention to the putative helix 8 of the receptor.     

Mechanism of the conversion of xanthine dehydrogenase to xanthine oxidase: identification of the two cysteine disulfide bonds and crystal structure of a non-convertible rat liver xanthine dehydrogenase mutant

Mammalian xanthine dehydrogenase can be converted to xanthine oxidase by modification of cysteine residues or by proteolysis of the enzyme polypeptide chain. Here we present evidence that the Cys(535) and Cys(992) residues of rat liver enzyme are indeed involved in the rapid conversion from the dehydrogenase to the oxidase. The purified mutants C535A and/or C992R were significantly resistant to conversion by incubation with 4,4'-dithiodipyridine, whereas the recombinant wild-type enzyme converted readily to the oxidase type, indicating that these residues are responsible for the rapid conversion. The C535A/C992R mutant, however, converted very slowly during prolonged incubation with 4,4'-dithiodipyridine, and this slow conversion was blocked by the addition of NADH, suggesting that another cysteine couple located near the NAD(+) binding site is responsible for the slower conversion. On the other hand, the C535A/C992R/C1316S and C535A/C992R/C1324S mutants were completely resistant to conversion, even on prolonged incubation with 4,4'-dithiodipyridine, indicating that Cys(1316) and Cys(1324) are responsible for the slow conversion. The crystal structure of the C535A/C992R/C1324S mutant was determined in its demolybdo form, confirming its dehydrogenase conformation.     

Fetal cells in mother rats contribute to the remodeling of liver and kidney after injury

Fetal microchimerism indicates a mixture of cells of maternal and fetal origin seen in maternal tissues during and after pregnancy. Controversy exists about whether persistent fetal microchimerism is related with some autoimmune disorders occurring during and after pregnancy. In the current experiment, an animal model in which EGFP positive cells were taken as fetal-origin cells was designed to detect the fetal microchimerism in various maternal organs. Ethanol drinking and gentamicin injection were adopted to induce liver and kidney injury simultaneously. EGFP positive cells were engrafted not only in the maternal circulation and bone marrow, but also in the liver and kidney as hepatocytes and tubular cells, respectively. These results indicate that fetal cells are engrafted to maternal hematopoietic system without apparent injury and they also contribute to the repairing process of maternal liver and kidney.     

Chemical rescue of a site-specific mutant of bacterial copper amine oxidase for generation of the topa quinone cofactor

The topa quinone (TPQ) cofactor of copper amine oxidase is produced by posttranslational modification of a specific tyrosine residue through the copper-dependent, self-catalytic process. We have site-specifically mutated three histidine residues (His431, His433, and His592) involved in binding of the copper ion in the recombinant phenylethylamine oxidase from Arthrobacter globiformis. The mutant enzymes, in which each histidine was replaced by alanine, were purified in the Cu/TPQ-free precursor form and analyzed for their Cu-binding and TPQ-generating activities by UV-visible absorption, resonance Raman, and electron paramagnetic resonance spectroscopies. Among the three histidine-to-alanine mutants, only H592A was found to show a weak activity to form TPQ upon aerobic incubation with Cu(2+) ions. Also for H592A, exogenous imidazole rescued binding of copper and markedly promoted the TPQ formation. Accommodation of a free imidazole molecule within the cavity created in the active site of H592A was suggested by X-ray crystallography. Although the TPQ cofactor in H592A mutant was readily reduced with substrate, its catalytic activity was very low even in the presence of imidazole. Combined with the crystal structures of the mutant enzymes, these results demonstrate the importance of the three copper-binding histidine residues for both TPQ biogenesis and catalytic activity, fine-tuning the position of the essential metal.     

Functional expression of rat ABCG2 on the luminal side of brain capillaries and its enhancement by astrocyte-derived soluble factor(s)

The purpose of the present study was to clarify the expression, transport properties and regulation of ATP-binding cassette G2 (ABCG2) transporter at the rat blood-brain barrier (BBB). The rat homologue of ABCG2 (rABCG2) was cloned from rat brain capillary fraction. In rABCG2-transfected HEK293 cells, rABCG2 was detected as a glycoprotein complex bridged by disulfide bonds, possibly a homodimer. The protein transported mitoxantrone and BODIPY-prazosin. In rat brain capillary fraction, rABCG2 protein was also detected as a glycosylated and disulfide-linked complex. Immunohistochemical analysis revealed that rABCG2 was localized mainly on the luminal side of rat brain capillaries, suggesting that rABCG2 is involved in brain-to-blood efflux transport. For the regulation study, conditionally immortalized rat brain capillary endothelial (TR-BBB13), astrocyte (TR-AST4) and pericyte (TR-PCT1) cell lines were used as an in vitro BBB model. Following treatment of TR-BBB13 cells with conditioned medium of TR-AST4 cells, the Ko143 (an ABCG2-specific inhibitor)-sensitive transport activity and rABCG2 mRNA level were significantly increased, whereas conditioned medium of TR-PCT1 cells had no effect. These results suggest that rat brain capillaries express functional rABCG2 protein and that the transport activity of the protein is up-regulated by astrocyte-derived soluble factor(s) concomitantly with the induction of rABCG2 mRNA.     

Tyrosine phosphorylation of BIT on photic stimulation in the rat retina

BIT is a transmembrane glycoprotein with three immunoglobulin-like domains in its extracellular region and tyrosine phosphorylation sites in its cytosolic region. We have previously shown that BIT was tyrosine phosphorylated in the hypothalamic suprachiasmatic nucleus in response to light exposure during the dark period, and suggested that it was involved in the light entrainment of the circadian clock. To further investigate the function of BIT in the nervous system, we examined the effect of photic stimulation on its tyrosine phosphorylation in the rat retina. It was found that the tyrosine phosphorylation level of BIT in the retina was higher in the light period than in the dark period. In addition, a light stimulation during the dark period resulted in a rapid phosphorylation of BIT and a subsequent association of BIT with SHP-2. The phosphorylation state was quickly reverted when the light was turned off. The light-dependent phosphorylation of BIT was also observed in isolated cultured retinas, and this was blocked by a specific Src-family inhibitor, PP-2. Immunohistochemical study showed that BIT was highly enriched in the inner and outer plexiform layers in the retina, where the immunoreactivity to anti-SHP-2 antibody was also detected. These results suggest that tyrosine phosphorylation of BIT is involved in neuronal transmission in the retina.     

Structural basis of the substrate-specific two-step catalysis of long chain fatty acyl-CoA synthetase dimer

Long chain fatty acyl-CoA synthetases are responsible for fatty acid degradation as well as physiological regulation of cellular functions via the production of long chain fatty acyl-CoA esters. We report the first crystal structures of long chain fatty acyl-CoA synthetase homodimer (LC-FACS) from Thermus thermophilus HB8 (ttLC-FACS), including complexes with the ATP analogue adenosine 5'-(beta,gamma-imido) triphosphate (AMP-PNP) and myristoyl-AMP. ttLC-FACS is a member of the adenylate forming enzyme superfamily that catalyzes the ATP-dependent acylation of fatty acid in a two-step reaction. The first reaction step was shown to propagate in AMP-PNP complex crystals soaked with myristate solution. Myristoyl-AMP was identified as the intermediate. The AMP-PNP and the myristoyl-AMP complex structures show an identical closed conformation of the small C-terminal domains, whereas the uncomplexed form shows a variety of open conformations. Upon ATP binding, the fatty acid-binding tunnel gated by an aromatic residue opens to the ATP-binding site. The gated fatty acid-binding tunnel appears only to allow one-way movement of the fatty acid during overall catalysis. The protein incorporates a hydrophobic branch from the fatty acid-binding tunnel that is responsible for substrate specificity. Based on these high resolution crystal structures, we propose a unidirectional Bi Uni Uni Bi Ping-Pong mechanism for the two-step acylation by ttLC-FACS.     

73-kDa molecular chaperone HSP73 is a direct target of antibiotic gentamicin

Although gentamicin (GM) has been used widely as an antibiotic, the specific binding protein of the drug has not yet been understood sufficiently. Here we show that GM specifically associates with the 73-kDa molecular chaperone HSP73 and reduces its chaperone activity in vitro. In the present study, we investigated GM-specific binding proteins using a GM-affinity column and porcine kidney cytosol. After washing the column, only the 73-kDa protein was eluted from the column by the addition of 10 mm GM. None of the other proteins were found in the eluant. Upon immunoblotting, the protein was identical to HSP73. Upon CD spectrum analysis, the binding of GM to HSP73 resulted in a conformational change in the protein. Although HSP73 prevents aggregation of unfolded rhodanese in vitro, the chaperone activity of HSP73 was suppressed in the presence of GM. Using limited proteolysis of HSP73 by TPCK-trypsin, the GM binding site is a COOH-terminal for one third of the protein known to be a peptide-binding domain. During immunohistochemistry, HSP73 and GM were co-localized in enlarged lysosomes of rat kidneys with GM-induced acute tubular injury in vivo. Our results suggest that the specific association between HSP73 and GM may reduce the chaperone activity of HSP73 in vitro and/or in vivo, and this may have an interaction with GM toxicity in kidneys with GM-induced acute tubular injury.     

Comparison of three different supercharging procedures in a rat skin flap model

A significant clinical problem in reconstructive surgery is partial loss of a pedicled flap. To resolve this problem, various methods of vascular augmentation have been developed; "supercharging" is one of those techniques. A new rat flap model was developed for investigation of the supercharging procedure, and the efficacy of the arterial supercharging method was examined. The purpose of this study was to investigate how an arterial supercharging procedure could generate large flap survival areas with different supercharging positions in rats. On the basis of the vascular anatomical features of rats, a circumferential skin flap from the lower abdomen to the back, measuring 4 x 12 cm, was marked. The flap was divided along the dorsal midline. Forty rats were divided into four experimental groups, as follows: group 1 (control), flaps based only on the deep circumflex iliac artery and vein; group 2, flaps supercharged with the ipsilateral superficial inferior epigastric artery; group 3, flaps supercharged with the contralateral superficial inferior epigastric artery; group 4, flaps supercharged with the contralateral deep circumflex iliac artery. On the fourth postoperative day, the flaps were evaluated with measurements of necrosis and survival areas. Microfil (Flow Tech, Inc., Carver, Mass.) was then injected manually throughout the body, and the vascular changes produced by supercharging were angiographically evaluated. Compared with group 1 (control), the flap survival areas were significantly greater in distally supercharged flaps in groups 3 and 4 (mean flap survival, 91.2 +/- 5.2 percent and 90.5 +/- 10.6 percent, respectively; p < 0.001) and in proximally supercharged flaps in group 2 (45.9 +/- 4.1 percent, p < 0.05). Angiographic assessment of the flaps that survived completely revealed marked dilation of the choke veins among the territories and reorientation of dilated veins along the axes of the flaps. This study suggests that distal arterial supercharging (contralateral superficial inferior epigastric artery or contralateral deep circumflex iliac artery) is more effective than proximal arterial supercharging (ipsilateral superficial inferior epigastric artery) in increasing flap survival. Although the rat skin flap may not be analogous to human flaps, distal arterial supercharging might have useful therapeutic potential in increasing flap survival in clinical practice.