Method for determining monohydroxybenzo[a]pyrene isomers using column-switching high-performance liquid chromatography

A method for determining monohydroxybenzo[a]pyrene (OHBaP) isomers using column-switching high-performance liquid chromatography with fluorescence detection was developed. Eleven of 12 isomers of OHBaP (all except 6-OHBaP) were separated on an alkylamide-type reversed-phase column and, via column-switching, on a beta-cyclodextrin-bonded silica gel column. The detection limits for the OHBaPs were in the range 0.3-8 pg/injection (S/N=3). By using this method, 1-, 3-, and 9-OHBaPs were identified as major metabolites of benzo[a]pyrene in vitro by human recombinant p450 1A1. The method was used to determine OHBaPs in the urine of a nonsmoker subject. After enzymatic hydrolysis of the conjugated metabolites by beta-glucuronidase/aryl sulfatase, the analytes were selectively adsorbed on blue rayon (a cellulose-supported copper phthalocyanine) from the urine matrix. Methanol as the eluting solvent from the rayon gave the best recoveries of OHBaPs and 1-hydroxypyrene (1-OHP) in the range of 91-103%, which was superior to that of the solid-phase extraction method. 1-OHP, a well-known biomarker of the exposure to polycyclic aromatic hydrocarbons, was simultaneously analyzed. Intra- and interday accuracy values for the determination of 3-OHBaP in 200 ml of urine were 95.5 and 100.9%, and those for 1-OHP were 96.4 and 103.6%, respectively. The intra- and interday precision values were 3.9 and 2.4% for 3-OHBaP and 2.4 and 3.2% for 1-OHP, respectively. In 11 kinds of isomers, only 3-OHBaP was detected in the human urine. Urinary concentration of 3-OHBaP was quantified at 0.5 ng/g creatinine concentration and the 3-OHBaP/1-OHP ratio was approximately 1/130.     

Alterations of structure and hydrolase activity of parkinsonism-associated human ubiquitin carboxyl-terminal hydrolase L1 variants

Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) is a neuron-specific ubiquitin recycling enzyme. A mutation at residue 93 and polymorphism at residue 18 within human UCH-L1 are linked to familial Parkinson's disease and a decreased Parkinson's disease risk, respectively. Thus, we constructed recombinant human UCH-L1 variants and examined their structure (using circular dichroism) and hydrolase activities. We confirmed that an I93M substitution results in a decrease in kcat (45.6%) coincident with an alteration in alpha-helical content. These changes may contribute to the pathogenesis of Parkinson's disease. In contrast, an S18Y substitution results in an increase in kcat (112.6%) without altering the circular dichroistic spectrum. These data suggest that UCH-L1 hydrolase activity may be inversely correlated with Parkinson's disease risk and that the hydrolase activity is protective against the disease. Furthermore, we found that oxidation of UCH-L1 by 4-hydroxynonenal, a candidate for endogenous mediator of oxidative stress-induced neuronal cell death, results in a loss of hydrolase activity. Taken together, these results suggest that further studies of altered UCH-L1 hydrolase function may provide new insights into a possible common pathogenic mechanism between familial and sporadic Parkinson's disease.     

Artificial chaperone-assisted refolding of bovine carbonic anhydrase using molecular assemblies of stimuli-responsive polymers

An artificial chaperone, which can decrease the protein aggregation and increase the reactivation yield of denatured protein in a fashion similar to natural chaperone, was newly developed using stimuli-responsive polymers. It has previously been reported that the addition of poly(propylene oxide)-phenyl-poly(ethylene glycol) (PPOn-Ph-PEG) with the unit number of PPO (n) 33 could enhance the refolding of bovine carbonic anhydrase (Kuboi et al. J. Chromatogr. B 2000, 243, 213). PPO-Ph-PEG with a large PPO chain (n = 50) was synthesized and the surface properties were characterized by both the relative fluorescence intensity of 1-anilino-8-naphthalene sulfonate (ANS) and the fluidity determined by diphenylhexatriene (DPH). The variation of ANS intensity and DPH fluidity is shown in a diagram as functions of temperature and polymer concentration. The high values of ANS intensity and fluidity of PPO50-Ph-PEG were obtained in a relatively wide conditional range (more than 0.08 mM and more than 15 degrees C) although the conditions showing the high values of PPO33-Ph-PEG were restricted (more than 0.1 mM and more than 40 degrees C). It was also found that molecular assemblies of PPOn-Ph-PEG with diameters of 7-18 nm were formed in the above conditions. On the basis of the surface properties of their polymer self-assemblies, the possibility of using them as an artificial chaperone was investigated. The effect of the addition of PPOn-Ph-PEG on the reactivation yield of a model protein, carbonic anhydrase from bovine (CAB), and the optical density of the solution was examined at various temperatures and concentrations. The reactivation yield of CAB was strongly enhanced and the aggregate formation (the optical density) was suppressed by adding PPOn-Ph-PEG in the above conditions, which show high ANS intensity and DPH fluidity. Especially in the presence of 0.1 mM PPO50-Ph-PEG, the reactivation yield of CAB reached approximately 100% at 40-55 degrees C. It was thus found that self-assemblies of the present polymer could be utilized as an artificial chaperone by selecting suitable stimuli conditions.     

Isolation,characterization, and cDNA sequence of a carotenoid binding protein from the silk gland of Bombyx mori larvae

A carotenoid binding protein (CBP) has been isolated from the silk glands of Bombyx mori larvae. The protein has an apparent molecular mass of 33 kDa and binds carotenoids in a 1:1 molar ratio. Lutein accounts for 90% of the bound carotenoids, whereas alpha-carotene and beta-carotene are minor components. Immunological analysis demonstrated the presence of CBP only in the yellow-colored tissues of the silk gland, midgut, testis, and ovary. Several phenotypes of B. mori mutants linked to carotenoid transport have been utilized to characterize CBP. The Y (yellow hemolymph) gene controls uptake of carotenoids from the midgut lumen into the midgut epithelium, and larvae with the +(Y) gene lack this property. Immunoblotting analysis confirmed the presence of CBP in mutants with the dominant Y gene only. Immunohistochemistry verified the localization of CBP in the villi of the midgut epithelium, indicating that CBP might be involved in absorption of carotenoids. A cDNA clone for CBP encoding a protein of 297 amino acids has been isolated from the B. mori silk gland cDNA library. The deduced amino acid sequence revealed that CBP is a novel member of the steroidogenic acute regulatory (StAR) protein family with its unique structural feature of a StAR-related lipid transfer domain, known to aid in lipid transfer and recognition. Lutein-binding capacity of the recombinant CBP (rCBP) determined by incubating rCBP with lutein followed by immunoprecipitation using anti-CBP IgG conjugated to protein A-Sepharose, demonstrated the formation of a lutein-rCBP complex. Sequence analyses coupled with binding specificity suggest that CBP is a new member of the StAR protein family that binds carotenoids rather than cholesterol.     

alpha-Tocopherol transfer protein expression in rat liver exposed to hyperoxia

-Tochopherol transfer protein ( TTP), a 32 kDa protein exclusively expressed in liver cytosol, has a high binding affinity for -tochopherol. The factors that regulate the expression of hepatic TTP are not clearly understood. In this study, we investigated whether or not exposure to hyperoxia (95% O 2 for 48 h) could alter the expression of hepatic TTP. We also examined the association between the expression of antioxidant enzymes (hepatic glutathione peroxidase (GPX) and Mn-superoxide dismutase (Mn-SOD)) and the expression of hepatic TTP. The levels of thiobarbituric acid-reactive substances (TBARS) in both plasma and liver were significantly higher after rats were exposed to hyperoxia for 48 h when compared with the levels in control rats. Northern blotting showed a decrease in the expression of TTP messenger RNA (mRNA) after hyperoxia, although the TTP protein level remained constant. Expression of Mn-SOD mRNA and protein, as well as the expression of GPX mRNA, were stable after hyperoxia. These findings indicate that mRNA for hepatic TTP, rather than Mn-SOD or GPX, may be highly responsive to oxidative stress.     

Transfer of the symbiotic plasmid from Rhizobium leguminosarum biovar trifolii to Agrobacterium tumefaciens

This study examined the symbiotic properties of Agrobacterium transconjugants isolated by transferring a Tn5-mob-marked derivative of the 315 kb megaplasmid pRt4Sa from Rhizobium leguminosarum bv. trifolii 4S (wild-type strain) to Agrobacterium tumefaciens A136 as the recipient. The genetic characteristics of the AT4S transconjugant strains were ascertained by random amplified polymorphic DNA (RAPD) analyses and Southern hybridization using Tn5-mob and nod genes as probes. Several of these AT4S transconjugants carrying pRt4Sa were able to nodulate roots of the normal legume host, white clover. In addition, some AT4S transconjugant strains were able to induce nodules on other leguminous plants, including alfalfa and hairy vetch. A characteristic bacteroid differentiation was observed in clover and alfalfa nodules induced by the AT4S-series strains, although nitrogen-fixing activity (acetylene reduction) was not found. Furthermore, strain H1R1, obtained by retracing transfer of the pRt4Sa::Tn5-mob from strain AT4Sa to strain H1 (pRt4Sa cured derivative of 4S), induced Fix(+) nodules on clover roots. These results indicate the evidence that only nod genes can be expressed in the Agrobacterium background.     

Selective separation process of proteins based on the heat stress-induced translocation across phospholipid membranes

Heating of several protein solutions at 40–47°C for 5–60 min in the presence of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) liposomes induced the translocation of β-galactosidase (β-gal), α-glucosidase (α-glu) and bovine carbonic anhydrase (CAB) from outer to inner aqueous phase across the liposome membrane. The translocated amounts of β-gal at various temperatures were maximized under suitable heating conditions (45°C, 30 min). Those of α-glu and CAB were maximized at 40–45 and 60°C, respectively. Each maximum value could be correlated with the corresponding local hydrophobicity of each protein evaluated by the aqueous two-phase partitioning method. The possibility to apply these heat-induced translocation phenomena to the bioseparation of proteins was successfully demonstrated for the model mixture solution of β-gal, α-glu and CAB.     

Tryptamine Induces Phosphoinositide Turnover and Modulates Adrenergic and Muscarinic Cholinergic Receptor Function in Cultured Cerebellar Granule Cells

Abstract: Tryptamine dose-dependently increased phosphoinositide (PI) hydrolysis by approximately fourfold in primary cultures of rat cerebellar granule cells (EC₅₀ = 56 µ M ). The PI response stimulated by tryptamine was dependent on the presence of extracellular Ca²⁺ and Na⁺. Tryptamine-induced PI breakdown could be partially inhibited by pretreatment with 4β-phorbol 12-myristate 13-acetate but not pertussis toxin. The presence of tryptamine markedly attenuated PI responses induced by norepinephrine (NE) and carbachol, with no apparent effect on the responses to 5-hydroxytryptamine and glutamate. The inhibition of NE- and carbachol-induced PI turnover by tryptamine was dose dependent with IC₅₀ values of ∼0.4 and ∼2.5 m M , respectively. Pretreatment of cells with tryptamine (0.5 m M ) also attenuated NE- and carbachol-induced PI turnover, but failed to affect 5-hydroxytryptamine- and glutamate-induced responses. Furthermore, ketanserin, atropine, and prazosin did not have any effect on inositol phosphate formation induced by tryptamine. These observations indicate that tryptamine markedly increased Ca²⁺- and Na⁺-dependent PI turnover in cerebellar neurons and selectively inhibited NE- and carbachol-induced PI hydrolysis.