Acetate Gradient Research Articles

Phosphorylation by Casein Kinase 1 Regulates Tonicity-induced Osmotic Response Element-binding Protein/Tonicity Enhancer-binding Protein Nucleocytoplasmic Trafficking

The osmotic response element-binding protein (OREBP), also known as tonicity enhancer-binding protein (TonEBP) or NFAT5, is the only known osmo-sensitive transcription factor that mediates cellular adaptations to extracellular hypertonic stress. Although it is well documented that the subcellular localization and transactivation activity of OREBP/TonEBP are tightly regulated by extracellular tonicity, the molecular mechanisms involved remain elusive. Here we show that nucleocytoplasmic trafficking of OREBP/TonEBP is regulated by the dual phosphorylation of Ser-155 and Ser-158. Alanine scanning mutagenesis revealed that Ser-155 is an essential residue that regulates OREBP/TonEBP nucleocytoplasmic trafficking. Tandem mass spectrometry revealed that Ser-155 and Ser-158 of OREBP/TonEBP are both phosphorylated in living cells under hypotonic conditions. In vitro phosphorylation assays further suggest that phosphorylation of the two serine residues proceeds in a hierarchical manner with phosphorylation of Ser-155 priming the phosphorylation of Ser-158 and that these phosphorylations are essential for nucleocytoplasmic trafficking of the transcription factor. Finally, we have shown that the pharmacological inhibition of casein kinase 1 (CK1) abolishes the phosphorylation of Ser-158 and impedes OREBP/TonEBP nuclear export and that recombinant CK1 phosphorylates Ser-158. Knockdown of CK1alpha1L, a novel isoform of CK1, inhibits hypotonicity-induced OREBP/TonEBP nuclear export. Together these data highlight the importance of Ser-155 and Ser-158 in the nucleocytoplasmic trafficking of OREBP/TonEBP and indicate that CK1 plays a major role in regulating this process.

HPLC Methods for Fucoxanthin Analysis in Dietary Supplements

Fucoxanthin (FX) is the second most prominent carotenoid in the earth's biomass. It is the primary carotenoid in some forms of brown algae. FX has been shown to reduce visceral adipose in mice and humans and is being incorporated into dietary supplements for weight loss. To examine the content of commercial FX products, we developed and validated Normal‐Phase and Reversed‐Phase HPLC methods to measure FX. Dietary supplements were purchased from local suppliers and over the internet. Samples were extracted into methanol prior to HPLC analysis. The NPLC was performed on a Diol column using a mobile phase of 6%IPA in hexane while monitoring 450nm. The RPLC was performed on a C30 column using a gradient of methanol, water, and ethyl acetate while monitoring 450nm. Samples tested contained various forms of seaweed or seaweed extracts. Products with stated FX content ranged from 250mcg to 5mg/unit. The products exhibit quite different carotenoid profiles and some do not meet label‐claims for FX. Due to current levels of obesity and increased use of thermogenic supplements, these methods will be useful to monitor the FX in current and future supplements.

Crystal structure of 2-benzyl-3-hydroxyisoindolin-1-one, C15H13NO2

C15H13NO2, monoclinic, P121/c1 (no. 14), a = 12.987(2) A, b = 7.691(1) A, c = 12.083(3) A, 3 = 96.970(4)°, V = 1198.0 A, Z = 4, Rgt(F) = 0.047, wRref(F) = 0.127, T = 153 K. Source of material The title compoundwas obtained from a two-step reaction. A free radical bromination was used to attach a bromide at the 4-H atom of phthalide, forming 3-bromophthalide (see CIF for details). 3Bromophthalide was reactedwith benzylamine in the presence of base: To a 100 mL round bottom flask with a magnetic stirbar, 3bromophthalide (2.24 g, 10.5mmol) was dissolved in acetonitrile (20mL). After stirring for 5min, diisopropylethylamine (3.16mL, 17.5 mmol) and benzylamine (1.26 mL, 11.4 mmol) were added. The reactionmixture was stirred at room temperature for 4 h. The solvent was then removed under conditions of reduced pressure. The crude residue was purified by silica gel chromatography, using hexane and ethyl acetate gradient. The product portion was collected and evaporated under reduced pressure (55 %). Yellow crystals were obtained by recrystallization from methanol solution. HNMR, CNMR, IR andMSdata are also available in the CIF. Discussion The title compound is the result of an unexpected ring-closing reaction, the analogous amide-aldehydewas the expected structure. TheN1,C10C8atoms of the fused rings are essentially co-planar with themaximum deviation from their least-squares plane being 0.017(1)A for theN1 atom; the carbonyl-O1 and benzyl-C9 atoms lying 0.020(1)A and –0.204(1)A out of this plane. The hydroxyl group lies to the opposite side of the fused ring system to that occupied by the benzyl residue. The molecule is bent at the benzyl-C9 atom as manifested in the dihedral angle of 84.38(6)° between the planes through the fused ring systemand the phenyl ring. The molecules are connected into a supramolecular chain along the [001] direction via O–H···O hydrogen bonds formed between the hydroxyl and carbonyl groups. The parameters associated with this interaction are d(O2–H···O1) = 1.87 A, d(O2···O1) = 2.707(2) A with an angle at H of 176° for i: x,1/2–y,–1/2+z. Z. Kristallogr. NCS 223 (2008) 25-26 / DOI 10.1524/ncrs.2008.0012 25 © by Oldenbourg Wissenschaftsverlag, Munchen Crystal: yellow prism, size 0.20 × 0.20 × 0.35 mm Wavelength: Mo K4 radiation (0.71070 A) *: 0.89 cm−1 Diffractometer, scan mode: Rigaku AFC12 & SATURN724, 6 2%max: 53.0° N(hkl)measured, N(hkl)unique: 8373, 2477 Criterion for Iobs, N(hkl)gt: Iobs > 2 #(Iobs), 2348 N(param)refined: 166 Programs: teXsan [1], SIR-92 [2], SHELXL-97 [3], ORTEP-II [4] Table 1. Data collection and handling.

Characterization of tRNA-dependent Peptide Bond Formation by MurM in the Synthesis of Streptococcus pneumoniae Peptidoglycan

MurM is an aminoacyl ligase that adds l-serine or l-alanine as the first amino acid of a dipeptide branch to the stem peptide lysine of the pneumococcal peptidoglycan. MurM activity is essential for clinical pneumococcal penicillin resistance. Analysis of peptidoglycan from the highly penicillin-resistant Streptococcus pneumoniae strain 159 revealed that in vivo and in vitro, in the presence of the appropriate acyl-tRNA, MurM(159) alanylated the peptidoglycan epsilon-amino group of the stem peptide lysine in preference to its serylation. However, in contrast, identical analyses of the penicillin-susceptible strain Pn16 revealed that MurM(Pn16) activity supported serylation more than alanylation both in vivo and in vitro. Interestingly, both MurM(Pn16) acylation activities were far lower than the alanylation activity of MurM(159). The resulting differing stem peptide structures of 159 and Pn16 were caused by the profoundly greater catalytic efficiency of MurM(159) compared with MurM(Pn16) bought about by sequence variation between these enzymes and, to a lesser extent, differences in the in vivo tRNA(Ala):tRNA(Ser) ratio in 159 and Pn16. Kinetic analysis revealed that MurM(159) acted during the lipid-linked stages of peptidoglycan synthesis, that the d-alanyl-d-alanine of the stem peptide and the lipid II N-acetylglucosaminyl group were not essential for substrate recognition, that epsilon-carboxylation of the lysine of the stem peptide was not tolerated, and that lipid II-alanine was a substrate, suggesting an evolutionary link to staphylococcal homologues of MurM such as FemA. Kinetic analysis also revealed that MurM recognized the acceptor stem and/or the TPsiC loop stem of the tRNA(Ala). It is anticipated that definition of the minimal structural features of MurM substrates will allow development of novel resistance inhibitors that will restore the efficacy of beta-lactams for treatment of pneumococcal infection.

Validation of a Micromethod for Quantification of Lutein and β‐Carotene in Olive Oil

A micromethod was developed and validated for the quantification of lutein and β‐carotene in olive oils. The compounds are liquid‐liquid extracted with n‐hexane and n,n–dimethylformamide (DMF), by vortex mixing and centrifugation. The n‐hexane phase retains β‐carotene, whereas the DMF phase retains lutein. This last fraction is concentrated and analyzed by RP‐HPLC/diode‐array, with a gradient of acetonitrile, water, and ethyl acetate, using β‐apo‐8′‐carotenal as I.S. β‐carotene is quantified by UV/Vis measurements at 454 nm, by the external standard method. The method shows appropriate inter‐ and intra‐day reproducibility, accuracy, and linearity for both compounds, allowing fast analysis of several samples simultaneously, with a relatively small consumption of organic solvents. The analytical methodology was applied to several samples of commercial Portuguese PDO olive oils.

Coupled Effect of Chemotaxis and Growth on Microbial Distributions in Organic-Amended Aquifer Sediments: Observations from Laboratory and Field Studies

The inter-relationship of growth and chemotactic response exhibited bytwo common soil-inhabiting bacteria was investigated to determine its impact on bacterial migration. Filter-chambers were used to simulate aquifer sediments characterized by vertical gradients of organic contaminants in both artificial groundwater flow systems in the laboratory and within the screened intervals of observation wells in a sandy aquifer. A labile model contaminant (acetate) was added to the top compartments of the three-part chambers, whereas bacteria with a demonstrated propensity to grow on and chemotactically respond to acetate were introduced to the lower compartments. The motility and chemotactic response of Pseudomonas putida F1 resulted in 40 to 110% greater abundances in the upper compartments and concomitant 22 to 70% depletions in the lower compartments relative to the nonchemotactic controls over 2 days. Bacteria were in greatest abundance within the sand plug that separated the upper and lower compartments where sharp acetate gradients induced a strong chemotactic response. This observation was consistent with predictions from a mathematical model. In agreement with the laboratory results, the down-well filter-chamber incubations with Pseudomonas stutzeri in the aquifer indicated that 91% fewer bacteria resided in the lower compartment than the control experiment without acetate at 15 h. The combination of chemotaxis and growth greatly accelerated the migration of bacteria toward and subsequent abundance at the higher acetate concentration.

Preparation, Characterization, and Evaluation of Liposomal Ferulic Acid In Vitro and In Vivo

In the present study, various gradients were evaluated for efficient loading of weak acid into liposomes. Several salt gradients showed efficient loading of ferulic acid (FA) into liposomes and the optimized conditions were established in calcium acetate gradient method to obtain 80.2 ± 5.2% entrapment efficiency (EE). Unilamellar vesicles were observed in micrographs and liposomal FA showed good stability. 80% of FA was released from liposomes within 5 h in vitro. There is a novel finding in this study: that drugs could be entrapped with a high solubility in the intraliposomal buffer in contrast to the low solubility in the extraliposomal buffer. The results of body distribution in rats indicated that liposomes could improve the body distribution of FA. For FA liposome, the concentration of FA in brain was two-fold higher than that of free FA. Liposomal FA was a promising approach to improve the body distribution of FA.

Amphipathic weak acid glucocorticoid prodrugs remote‐loaded into sterically stabilized nanoliposomes evaluated in arthritic rats and in a Beagle dog: A novel approach to treating autoimmune arthritis

The use of glucocorticoids (GCs) in rheumatoid arthritis is limited by side effects related to unfavorable pharmacokinetics and biodistribution. Liposomal GC formulations have been studied since the 1970s in an attempt to overcome this obstacle, but none has entered clinical use. We undertook this study to determine whether a novel approach could overcome the limitations that have thus far prevented the clinical use of these formulations: low drug:lipid ratio, low encapsulation efficiency, and lack of controlled release. We used approximately 80-nm sterically stabilized (pegylated) nanoliposomes (NSSLs), which were remote-loaded with an amphipathic weak acid GC (such as methyl prednisolone hemisuccinate) utilizing an intraliposome (aqueous compartment)-high/extraliposome (bulk medium)-low transmembrane calcium acetate gradient. This unique method actually "traps" the GC in the liposomal aqueous phase as a calcium-GC precipitate. Our liposome formulation exhibited high encapsulation efficiency (94%) and a high drug:lipid mole ratio (0.41) and demonstrated controlled release of the encapsulated GC during systemic circulation and in inflamed paws in rats with adjuvant-induced arthritis. In addition, both in arthritic rats and in a Beagle dog, we showed the pharmacokinetic advantage of using liposomes as GC carriers. Finally, we demonstrated the superior therapeutic efficacy of our liposome formulation over that of free GCs in arthritic rats, both in early and in peak disease stages. Amphipathic weak acid GCs remote-loaded into approximately 80-nm NSSLs overcome past limitations of liposomal GC formulations. The unique loading method, which also leads to controlled release, improves the therapeutic effect both systemically and locally. Such a development has great potential for improving GC therapy.

Functional Characterization of the Atypical Hsp70 Subunit of Yeast Ribosome-associated Complex

Eukaryotic ribosomes carry a stable chaperone complex termed ribosome-associated complex consisting of the J-domain protein Zuo1 and the Hsp70 Ssz1. Zuo1 and Ssz1 together with the Hsp70 homolog Ssb1/2 form a functional triad involved in translation and early polypeptide folding processes. Strains lacking one of these components display slow growth, cold sensitivity, and defects in translational fidelity. Ssz1 diverges from canonical Hsp70s insofar that neither the ability to hydrolyze ATP nor binding to peptide substrates is essential in vivo. The exact role within the chaperone triad and whether or not Ssz1 can hydrolyze ATP has remained unclear. We now find that Ssz1 is not an ATPase in vitro, and even its ability to bind ATP is dispensable in vivo. Furthermore, Ssz1 function was independent of ribosome-associated complex formation, indicating that Ssz1 is not merely a structural scaffold for Zuo1. Finally, Ssz1 function in vivo was inactivated when both nucleotide binding and Zuo1 interaction via the C-terminal domain were disrupted in the same mutant. The two domains of this protein thus cooperate in a way that allows for severe interference in either but not in both of them.

LC-MS Study To Reduce Ion Suppression and To Identify N-Lactoylguanosine 5‘-Monophosphate in Bonito: A New Umami Molecule?

In this study a specific taste modulating flavor ingredient, N-lactoylguanosine 5'-monophosphate (N-lactoyl GMP), was determined in bonito (Japanese, Katsuobushi, dried fermented skipjack) and in powdered bonito using liquid chromatography-electrospray ionization (+) mass spectrometry-mass spectrometry (LC-ESI(+)-MS/MS) with a methanol/ammonium acetate or formate gradient. Furthermore, the influence of ion suppression due to sample matrix effect was investigated and was found to substantially influence the total MS response of N-lactoyl GMP; by adjusting the LC conditions the response could be approximately 5-fold-enhanced. The N-lactoyl GMP concentrations in different types of bonito products were between 0.2 and 2.4 microg/g.

Diversity of Translation Start Sites May Define Increased Complexity of the Human Short ORFeome

Our previous proteomics analysis of small proteins expressed in human K562 cells provided the first direct evidence of translation of upstream ORFs in human full-length cDNAs (Oyama, M., Itagaki, C., Hata, H., Suzuki, Y., Izumi, T., Natsume, T., Isobe, T., and Sugano, S. (2004) Analysis of small human proteins reveals the translation of upstream open reading frames of mRNAs. Genome Res. 14, 2048-2052). In the present study, we performed an in-depth proteomics analysis of human K562 and HEK293 cells using a two-dimensional nano-liquid chromatography-tandem mass spectrometry system. The results led to the identification of eight protein-coding regions besides 197 small proteins with a theoretical mass less than 20 kDa that were already annotated coding sequences in the curated mRNA database. In addition to the upstream ORFs in the presumed 5'-untranslated regions of mRNAs, bioinformatics analysis based on accumulated 5'-end cDNA sequence data provided evidence of novel short coding regions that were likely to be translated from the upstream non-AUG start site or from the new short transcript variants generated by utilization of downstream alternative promoters. Protein expression analysis of the GRINL1A gene revealed that translation from the most upstream start site occurred on the minor alternative splicing transcript, whereas this initiation site was not utilized on the major mRNA, resulting in translation of the downstream ORF from the second initiation codon. These findings reveal a novel post-transcriptional system that can augment the human proteome via the alternative use of diverse translation start sites coupled with transcriptional regulation through alternative promoters or splicing, leading to increased complexity of short protein-coding regions defined by the human transcriptome.

Surface Modification of RGD-Liposomes for Selective Drug Delivery to Monocytes/Neutrophils in Brain

In the present study, RGD peptide was coupled with ferulic acid (FA) liposomes for binding to monocytes and neutrophils in peripheral blood for brain targeting in response to leukocyte recruitment. Cholesterol (Ch) was esterified with succinic anhydride to introduce a carboxylic end group (Ch-COOH). Soybean phosphatidylcholine, cholesterol and Ch-COOH were in a molar ratio of 1 : 0.23 : 0.05. FA was loaded into liposomes with 80.2+/-5.2% entrapment efficiency (EE) using a calcium acetate gradient method since it was difficult to load FA by other methods. RGD peptide was a novel compound coupled with Ch-COOH via carbodiimide and N-hydroxysulfosuccinimide. The results of the in vitro flow cytometric study showed that RGD conjugation liposomes (RGD-liposomes) could bind to monocytes/neutrophils efficiently. The rats were subjected to intrastriatal microinjections of 100 microl of human recombinant IL-1beta to produce brain inflammation and subsequently sacrificed after 15, 30, 60 and 120 min of administration of three formulations (FA solution, FA liposome, RGD-coated FA liposome). The body distribution results showed that RGD-liposomes could be directed to the target site, i.e. the brain, by cell selectivity in case of an inflammatory response. For RGD coated liposomes, the concentration of FA in brain was 6-fold higher than that of FA solution and 3-fold higher than that of uncoated liposomes. MTT assay and flow cytometry were used in the pharmacodynamic studies where it was found that FA liposomes exhibited greater antioxidant activity to FA solution on U937 cell.

An ent-kaurane diterpene from Mikania laevigata (Asteraceae)

The genus Mikania contains more than 450 species and around 150 of them are found in Brazil [1]. This genus is divided in two groups of species, one of them produces preferently sesquiterpene lactones and the other, diterpenic acids. The late compounds seem to play an important role in defense system of plants [2]. Mikania laevigata Schultz Bip. ex Baker („guaco“) is the most abundant Mikania species in Southern Brazil [3]. The leaves are used mainly to treat respiratory diseases, but antiseptic, antiasthmatic and antirheumatic properties have been described. The characteristic smell of Mikania species is due to coumarins and some of their properties are also attributed to these compounds [4]. The plant material was cultivated at Federal University of Santa Catarina. The dried and powdered leaves (281g) were macerated with ethanol during 7 days. The crude extract was treated with MeOH:H2O (9:1), and extracted with petrol (PE), CH2Cl2, ethyl acetate and n-butanol. PE fraction was fractionated by flash chromatography (gradient of PE:CH2Cl2) followed by silica gel column chromatography (PE:ethyl acetate gradient) and Sephadex LH-20 (MeOH), yielding 21mg of compound 1. The NMR-H1, NMR-C13, IR and UV data suggested compound 1 to be ent-15β-benzoyloxy-kaur-16(17)-en-19-oic acid. This compound has been isolated also from other Mikania species [5,6], but it has never been found in M. laevigata, therefore being a new compound of this species.

Accurate Determination of Human Serum Transferrin Isoforms: Exploring Metal-Specific Isotope Dilution Analysis as a Quantitative Proteomic Tool

Carbohydrate-deficient transferrin (CDT) measurements are considered a reliable marker for chronic alcohol consumption, and its use is becoming extensive in forensic medicine. However, CDT is not a single molecular entity but refers to a group of sialic acid-deficient transferrin isoforms from mono- to trisialotransferrin. Thus, the development of methods to analyze accurately and precisely individual transferrin isoforms in biological fluids such as serum is of increasing importance. The present work illustrates the use of ICPMS isotope dilution analysis for the quantification of transferrin isoforms once saturated with iron and separated by anion exchange chromatography (Mono Q 5/50) using a mobile phase consisting of a gradient of ammonium acetate (0-250 mM) in 25 mM Tris-acetic acid (pH 6.5). Species-specific and species-unspecific spikes have been explored. In the first part of the study, the use of postcolumn addition of a solution of 200 ng mL(-1) isotopically enriched iron (57Fe, 95%) in 25 mM sodium citrate/citric acid (pH 4) permitted the quantification of individual sialoforms of transferrin (from S2 to S5) in human serum samples of healthy individuals as well as alcoholic patients. Second, the species-specific spike method was performed by synthesizing an isotopically enriched standard of saturated transferrin (saturated with 57Fe). The characterization of the spike was performed by postcolumn reverse isotope dilution analysis (this is, by postcolumn addition of a solution of 200 ng mL(-1) natural iron in sodium citrate/citric acid of pH 4). Also, the stability of the transferrin spike was tested during one week with negligible species transformation. Finally, the enriched transferrin was used to quantify the individual isoforms in the same serum samples obtaining results comparative to those of postcolumn isotope dilution and to those previously published in the literature, demonstrating the suitability of both strategies for quantitative transferrin isoform determination in real samples.

Trypanocidal properties of <i>Terminalia ivorensis</i> A. Chev. (Combretaceae)

Human African Trypanosomiasis (sleeping sickness) cause considerable mortality and morbidity throughout the world with Trypansoma brucei, T. gambiense and T. brucei rhodesiense as the major causative agents. The Terminalia ivorensis gradient extracts were tested against T. brucei rhodesiense parasites and the E.D50 values of 11.11 and 12.32 µg/mL were obtained for the ethyl acetate and ethanolic gradient extracts respectively. The column fractions of the ethyl acetate fraction gave E.D50 value between 1.98 and 5.46 µg/mL for the very active fractions indicating prospect for the development of potential trypanocidal agent from the plant.

Behavioral effects of a neurotoxic compound isolated from Clibadium surinamense L (Asteraceae)

Clibadium surinamense L, popularly known as cunambi, is a native plant from the Northern region of Brazil illegally used for predatory fishing. Previous results from our laboratory have demonstrated that the oral treatment of mice with the ethanolic extract (EE) of the leaves of the plant induced generalized tonic–clonic seizures followed by death within 30 min. The aims of the present paper were to characterize the convulsant effect of the hexanic extract (HE) of the stems and leaves of C. surinamense and, by bioguided purification, to identify the active principle and its mechanism of action. The leaves and stems were extracted with hexane (100 g/L) in Soxhlet for 36 h (yield of 2.4%), the solvent was evaporated and the powder dissolved in 1.5% saline/Tween 80. Male mice (30–35 g) treated with HE (22.5–360 mg/kg, p.o.) showed behavioral alterations consistent with CNS stimulation. The intensity and duration of the effect were proportional to the administered doses. The behavioral alterations, measured with a graded score of seizure severity, revealed that pretreatment with carbamazepine (30 mg/kg, i.p., 60 min) or phenytoin (50 mg/kg, i.p., 30 min) did not alter the HE convulsive effect. In contrast, phenobarbital (30 mg/kg, i.p., 60 min) or diazepam (2 mg/kg, i.p., 30 min) reduced the HE effect, increasing the ED 50 for clonic seizures from 64.4 to 89.8 mg/kg and 168.9 mg/kg, respectively. Purification of the HE in a silica gel column eluted with a hexane/ethyl acetate gradient yielded a single fraction with convulsant effect in which cunaniol acetate was identified by 1H NMR as the main active compound. These results indicated that inhibition of GABAergic transmission by cunaniol acetate might be responsible for the convulsant effects of C. surinamense L in mice, but do not exclude a direct cunaniol action labilizing neuronal excitability.

Improvement in HPLC separation of porphyrin isomers and application to biochemical diagnosis of porphyrias

Identification of porphyrias relies on the measurement of different porphyrins in urine, feces and plasma. Separation of porphyrin isomers is essential for the differential diagnosis of some porphyrias. Separation of naturally occurring porphyrins was achieved on a Chromolith RP-18 column with fluorimetric detection using a methanol/ammonium acetate gradient mobile phase. Fecal and plasma porphyrins were extracted with acetonitrile and water at different pH values. Eight porphyrins including protoporphyrin eluted within 20 min with good resolution of each of the I and III positional isomer pairs for standards, urine and plasma, and within 50 min for feces. Improvement of the extraction method for fecal and plasmatic porphyrins resulted in high recovery (up to 89%) and reliable quantification of protoporphyrin. The present RP-HPLC method is specific and efficient for routine analysis of porphyrins in human urine, feces and plasma.

Determination of schizandrin in rat plasma by high-performance liquid chromatography–mass spectrometry and its application in rat pharmacokinetic studies

A sensitive liquid chromatography-mass spectrometric (LC/MS) method for the quantification of schizandrin in rat plasma was developed and validated after solid-phase extraction (SPE). Chromatographic separation was achieved on a reversed-phase Shimadzu C(18) column with the mobile phase of acetonitrile-sodium acetate (10 micromol/L) and step gradient elution resulted in a total run time of about 11.7 min. The analytes were detected using an electrospray positive ionization mass spectrometry in the selected ion monitoring (SIM) mode. A good linear relationship was obtained in the concentration range studied (0.005-2.000 microg/mL) (r=0.9999). Lower limit of quantification (LLOQ) was 5 ng/mL and the lower limit of detection (LLOD) was 2 ng/mL using 100 microL plasma sample. Average recoveries ranged from 75.85 to 88.51% in plasma at the concentrations of 0.005, 0.100 and 1.000 microg/mL. Intra- and inter-day relative standard deviations were 5.95-12.93% and 3.87-14.53%, respectively. This method was successfully applied for the pharmacokinetic studies in rats.

Lecithin:Retinol Acyltransferase Is Responsible for Amidation of Retinylamine, a Potent Inhibitor of the Retinoid Cycle

Lecithin:retinol acyltransferase (LRAT) catalyzes the transfer of an acyl group from the sn-1 position of phosphatidylcholine to all-trans-retinol (vitamin A) and plays an essential role in the regeneration of visual chromophore as well as in the metabolism of vitamin A. Here we demonstrate that retinylamine (Ret-NH2), a potent and selective inhibitor of 11-cis-retinal biosynthesis (Golczak, M., Kuksa, V., Maeda, T., Moise, A. R., and Palczewski, K. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 8162-8167), is a substrate for LRAT. LRAT catalyzes the transfer of the acyl group onto Ret-NH2 leading to the formation of N-retinylpalmitamide, N-retinylstearamide, and N-retinylmyristamide with a ratio of 15:6:2, respectively. The presence of N-retinylamides was detected in vivo in mice supplemented with Ret-NH2. N-Retinylamides are thus the main metabolites of Ret-NH2 in the liver and the eye and can be mobilized by hydrolysis/deamidation back to Ret-NH2. Using two-photon microscopy and the intrinsic fluorescence of N-retinylamides, we showed that newly formed amides colocalize with the retinyl ester storage particles (retinosomes) in the retinal pigment epithelium. These observations provide new information concerning the substrate specificity of LRAT and explain the prolonged effect of Ret-NH2 on the rate of 11-cis-retinal recovery in vivo.

The Role of Mannosylated Enzyme and the Mannose Receptor in Enzyme Replacement Therapy

Lysosomal acid lipase (LAL) is the critical enzyme for the hydrolysis of triglycerides (TGs) and cholesteryl esters (CEs) in lysosomes. LAL defects cause Wolman disease (WD) and CE storage disease (CESD). An LAL null (lal-/-) mouse model closely mimics human WD/CESD, with hepatocellular, Kupffer cell and other macrophage, and adrenal cortical storage of CEs and TGs. The effect on the cellular targeting of high-mannose and complex oligosaccharide-type oligosaccharide chains was tested with human LAL expressed in Pichia pastoris (phLAL) and CHO cells (chLAL), respectively. Only chLAL was internalized by cultured fibroblasts, whereas both chLAL and phLAL were taken up by macrophage mannose receptor (MMR)-positive J774E cells. After intraperitoneal injection into lal-/- mice, phLAL and chLAL distributed to macrophages and macrophage-derived cells of various organs. chLAL was also detected in hepatocytes. Ten injections of either enzyme over 30 d into 2- and 2.5-mo-old lal-/- mice produced normalization of hepatic color, decreased liver weight (50%-58%), and diminished hepatic cholesterol and TG storage. Lipid accumulations in macrophages were diminished with either enzyme. Only chLAL cleared lipids in hepatocytes. Mice double homozygous for the LAL and MMR deficiences (lal-/-;MMR-/-) showed phLAL uptake into Kupffer cells and hepatocytes, reversal of macrophage histopathology and lipid storage in all tissues, and clearance of hepatocytes. These results implicate MMR-independent and mannose 6-phosphate receptor-independent pathways in phLAL uptake and delivery to lysosomes in vivo. In addition, these studies show specific cellular targeting and physiologic effects of differentially oligosaccharide-modified human LALs mediated by MMR and that lysosomal targeting of mannose-terminated glycoproteins occurs and storage can be eliminated effectively without MMR.