The Non-Mendelian Green Cotyledon Gene in Soybean Encodes a Small Subunit of Photosystem II.

Chlorophyll degradation plays important roles in leaf senescence including regulation of degradation of chlorophyll-binding proteins. Although most genes encoding enzymes of the chlorophyll degradation pathway have been identified, the regulation of their activity has not been fully understood. Green cotyledon mutants in legume are stay-green mutants, in which chlorophyll degradation is impaired during leaf senescence and seed maturation. Among them, the soybean (Glycine max) green cotyledon gene cytG is unique because it is maternally inherited. To isolate cytG, we extensively sequenced the soybean chloroplast genome, and detected a 5-bp insertion causing a frame-shift in psbM, which encodes one of the small subunits of photosystem II. Mutant tobacco plants (Nicotiana tabacum) with a disrupted psbM generated using a chloroplast transformation technique had green senescent leaves, confirming that cytG encodes PsbM. The phenotype of cytG was very similar to that of mutant of chlorophyll b reductase catalyzing the first step of chlorophyll b degradation. In fact, chlorophyll b-degrading activity in dark-grown cytG and psbM-knockout seedlings was significantly lower than that of wild-type plants. Our results suggest that PsbM is a unique protein linking photosynthesis in presenescent leaves with chlorophyll degradation during leaf senescence and seed maturation. Additionally, we discuss the origin of cytG, which may have been selected during domestication of soybean.

19F Nuclear Magnetic Resonance Spectrometric Determination of the Partition Coefficients of Flutamide and Nilutamide (Antiprostate Cancer Drugs) in a Lipid Nano-Emulsion and Prediction of Its Encapsulation Efficiency for the Drugs.

To design a useful lipid drug carrier having a high encapsulation efficiency (EE%) for the antiprostate cancer drugs flutamide (FT) and nilutamide (NT), a lipid nano-emulsion (LNE) was prepared with soybean oil (SO), phosphatidylcholine (PC), and sodium palmitate, and the partition coefficients (K ps) of the drugs for the LNE were determined by 19F nuclear magnetic resonance (NMR) spectrometry. The 19F NMR signal of the trifluoromethyl group of both drugs showed a downfield shift from an internal standard (trifluoroethanol) and broadening according to the increase in the lipid concentration due to their interaction with LNE particles. The difference in the chemical shift (Δδ) of each drug caused by the addition of LNE was measured under different amounts of LNE, and the K p values were calculated from the Δδ values. The results showed that FT has higher lipophilicity than NT. The total lipid concentration (SO + PC) required to encapsulate each drug into LNE with an EE% of more than 95% was calculated from the K p values as 93.3 and 189.9 mmol/L for FT and NT, respectively. For an LNE prepared with the total lipid concentration of 215 mmol/L, the predicted EE% values were 98 and 96% for FT and NT, respectively, while the experimental EE% values determined by a centrifugation method were approximately 99% for both drugs. Thus, the 19F NMR spectrometric method is a useful technique to obtain the K p values of fluorinated drugs and thereby predict the theoretical lipid concentrations and prepare LNEs with high EE% values.

A green-cotyledon/stay-green mutant exemplifies the ancient whole-genome duplications in soybean.

The recent whole-genome sequencing of soybean (Glycine max) revealed that soybean experienced whole-genome duplications 59 million and 13 million years ago, and it has an octoploid-like genome in spite of its diploid nature. We analyzed a natural green-cotyledon mutant line, Tenshin-daiseitou. The physiological analysis revealed that Tenshin-daiseitou shows a non-functional stay-green phenotype in senescent leaves, which is similar to that of the mutant of Mendel's green-cotyledon gene I, the ortholog of SGR in pea. The identification of gene mutations and genetic segregation analysis suggested that defects in GmSGR1 and GmSGR2 were responsible for the green-cotyledon/stay-green phenotype of Tenshin-daiseitou, which was confirmed by RNA interference (RNAi) transgenic soybean experiments using GmSGR genes. The characterized green-cotyledon double mutant d1d2 was found to have the same mutations, suggesting that GmSGR1 and GmSGR2 are D1 and D2. Among the examined d1d2 strains, the d1d2 strain K144a showed a lower Chl a/b ratio in mature seeds than other strains but not in senescent leaves, suggesting a seed-specific genetic factor of the Chl composition in K144a. Analysis of the soybean genome sequence revealed four genomic regions with microsynteny to the Arabidopsis SGR1 region, which included the GmSGR1 and GmSGR2 regions. The other two regions contained GmSGR3a/GmSGR3b and GmSGR4, respectively, which might be pseudogenes or genes with a function that is unrelated to Chl degradation during seed maturation and leaf senescence. These GmSGR genes were thought to be produced by the two whole-genome duplications, and they provide a good example of such whole-genome duplication events in the evolution of the soybean genome.

Knockdown of the 7S globulin subunits shifts distribution of nitrogen sources to the residual protein fraction in transgenic soybean seeds.

KEY MESSAGE: A platform of gene silencing by amiRNA had been established in fertile transgenic soybean. We demonstrated that knockdown of storage protein shifted the distribution of nitrogen sources in soybean seeds. Artificial microRNAs (amiRNAs) were designed using the precursor sequence of the endogenous soybean (Glycine max L. Merrill) miRNA gma-miR159a and expressed in transgenic soybean plants to suppress the biosynthesis of 7S globulin, which is one of the major storage proteins. Seed-specific expression of these amiRNAs (amiR-7S) resulted in a strong suppression of 7S globulin subunit genes and decreased accumulation of the 7S globulin subunits in seeds. Thus, the results demonstrate that a platform for gene silencing by amiRNA was first developed in fertile transgenic soybean plants. There was no difference in nitrogen, carbon, and lipid contents between amiR-7S and control seeds. Four protein fractions were collected from defatted mature seeds on the basis of solubility at different pH to examine the distribution of nitrogen sources and compensatory effects. In the whey and lipophilic fractions, nitrogen content was similar in amiR-7S and control seeds. Nitrogen content was significantly decreased in the major soluble protein fraction and increased in the residual fraction (okara) of the amiR-7S seeds. Amino acid analysis revealed that increased nitrogen compounds in okara were proteins or peptides rather than free amino acids. Our study indicates that the decrease in 7S globulin subunits shifts the distribution of nitrogen sources to okara in transgenic soybean seeds.

Genetic variation of γ-tocopherol methyltransferase gene contributes to elevated α-tocopherol content in soybean seeds.

BACKGROUND: Improvement of α-tocopherol content is an important breeding aim to increase the nutritional value of crops. Several efforts have been conducted to improve the α-tocopherol content in soybean [Glycine max (L.) Merr.] through transgenic technology by overexpressing genes related to α-tocopherol biosynthesis or through changes to crop management practices. Varieties with high α-tocopherol content have been identified in soybean germplasms. The heritability of this trait has been characterized in a cross between high α-tocopherol variety Keszthelyi Aproszemu Sarga (KAS) and low α-tocopherol variety Ichihime. In this study, the genetic mechanism of the high α-tocopherol content trait of KAS was elucidated. RESULTS: Through QTL analysis and fine mapping in populations from a cross between KAS and a Japanese variety Ichihime, we identified γ-TMT3, which encodes γ-tocopherol methyltransferase, as a candidate gene responsible for high α-tocopherol concentration in KAS. Several nucleotide polymorphisms including two nonsynonymous mutations were found in the coding region of γ-TMT3 between Ichihime and KAS, but none of which was responsible for the difference in α-tocopherol concentration. Therefore, we focused on transcriptional regulation of γ-TMT3 in developing seeds and leaves. An F5 line that was heterozygous for the region containing γ-TMT3 was self-pollinated. From among the progeny, plants that were homozygous at the γ-TMT3 locus were chosen for further evaluation. The expression level of γ-TMT3 was higher both in developing seeds and leaves of plants homozygous for the γ-TMT3 allele from KAS. The higher expression level was closely correlated with high α-tocopherol content in developing seeds. We generated transgenic Arabidopsis plants harboring GUS gene under the control of γ-TMT3 promoter from KAS or Ichihime. The GUS activity assay showed that the activity of γ-TMT3 promoter from KAS was higher than that of Ichihime. CONCLUSIONS: The genetic variation in γ-TMT3, which plays a major role in determining α-tocopherol concentration, provides significant information about the regulation of tocopherol biosynthesis in soybean seeds. This knowledge will help breeding programs to develop new soybean varieties with high α-tocopherol content.

The soybean stem growth habit gene Dt1 is an ortholog of Arabidopsis TERMINAL FLOWER1.

Classical genetic analysis has revealed that the determinate habit of soybean (Glycine max) is controlled by a recessive allele at the determinate stem (Dt1) locus. To dissect the molecular basis of the determinate habit, we isolated two orthologs of pea (Pisum sativum) TERMINAL FLOWER1a, GmTFL1a and GmTFL1b, from the soybean genome. Mapping analysis indicated that GmTFL1b is a candidate for Dt1. Despite their high amino acid identity, the two genes had different transcriptional profiles. GmTFL1b was expressed in the root and shoot apical meristems (SAMs), whereas GmTFL1a was mainly expressed in immature seed. The GmTFL1b transcript accumulated in the SAMs during early vegetative growth in both the determinate and indeterminate lines but thereafter was abruptly lost in the determinate line. Introduction of the genomic region of GmTFL1b from the indeterminate line complemented the stem growth habit in the determinate line: more nodes were produced, and flowering in the terminal raceme was delayed. The identity between Dt1 and GmTFL1b was also confirmed with a virus-induced gene silencing experiment. Taken together, our data suggest that Dt1 encodes the GmTFL1b protein and that the stem growth habit is determined by the variation of this gene. The dt1 allele may condition the determinate habit via the earlier loss in GmTFL1b expression concomitant with floral induction, although it functions normally under the noninductive phase of flowering. An association test of DNA polymorphisms with the stem growth habit among 16 cultivars suggested that a single amino acid substitution in exon 4 determines the fate of the SAM after floral induction.

Manipulation of amino acid composition in soybean seeds by the combination of deregulated tryptophan biosynthesis and storage protein deficiency.

The ability of genetic manipulation to yield greatly increased concentrations of free amino acids (FAAs) in seeds of soybean was evaluated by introduction of a feedback-insensitive mutant enzyme of tryptophan (Trp) biosynthesis into two transformation-competent breeding lines deficient in major seed storage proteins. The storage protein-deficient lines exhibited increased accumulation of certain other seed proteins as well as of FAAs including arginine (Arg) and asparagine in mature seeds. Introduction of the gene for a feedback-insensitive mutant of an alpha subunit of rice anthranilate synthase (OASA1D) into the two high-FAA breeding lines by particle bombardment resulted in a >10-fold increase in the level of free Trp in mature seeds compared with that in nontransgenic seeds. The amount of free Trp in these transgenic seeds was similar to that in OASA1D transgenic seeds of the wild-type cultivar Jack. The composition of total amino acids in seeds of the high-FAA breeding lines remained largely unaffected by the expression of OASA1D with the exception of an increase in the total Trp content. Our results therefore indicate that the extra nitrogen resource originating from storage protein deficiency was used exclusively for the synthesis of inherent alternative nitrogen reservoirs such as free Arg and not for deregulated Trp biosynthesis conferred by OASA1D. The intrinsic null mutations responsible for storage protein deficiency and the OASA1D transgene affecting Trp content were thus successfully combined and showed additive effects on the amino acid composition of soybean seeds.

A novel major quantitative trait locus controlling seed development at low temperature in soybean (Glycine max).

Low temperature is among the critical environmental factors that limit soybean production. To elucidate the genetic basis for chilling tolerance and identify useful markers, we conducted quantitative trait loci (QTL) analysis of seed-yielding ability at low temperature in soybean (Glycine max), using artificial climatic environments at usual and low temperatures and recombinant inbred lines derived from a cross between two contrasting cultivars in terms of chilling tolerance. We identified a QTL of a large effect (LOD > 15, r (2) > 0.3) associated with seed-yielding ability only at low temperature. The QTL was mapped near marker Sat_162 on linkage group A2, where no QTL for chilling tolerance has previously been identified. The tolerant genotype did not increase the pod number but maintained the seed number per pod and single seed weight, namely, the efficiency of seed development at low temperature. The effect of the QTL was confirmed in a segregating population of heterogeneous inbred families, which provided near-isogenic lines. The genomic region containing the QTL also influenced the node and pod numbers regardless of temperature condition, although this effect was not primarily associated with chilling tolerance. These results suggest the presence of a new major genetic factor that controls seed development specifically at low temperature. The findings will be useful for marker-assisted selection as well as for understanding of the mechanism underlying chilling tolerance in reproductive organs.

Genetic mapping of Cgdef gene controlling accumulation of 7S globulin (beta-conglycinin) subunits in soybean seeds.

Soy protein consists of mainly 7S globulin (beta-conglycinin) and 11S globulin (glycinin). The 7S globulin exerts favorable and unfavorable effects on human nutrition, food processing, and human health. Therefore, it is important for the improvement of the soy protein to control the content of 7S globulin. A mutant line lacking the 7S globulin was induced by gamma-ray irradiation, and the deficiency is controlled by a single recessive gene, cgdef. The Cgdef gene, despite its potential for improvement of the soy protein, has not been assigned to a linkage group of a soybean genetic map. We crossed "Mo-shi-dou Gong 503" with plants homozygous or heterozygous for the Cgdef allele and screened an F2 mapping population that segregated with the cgdef allele to locate the Cgdef gene on a soybean genetic map. By linkage analysis, we assigned the Cgdef gene to chromosome 19 at the position between the Satt523 and Sat_388 simple sequence repeat (SSR) markers. Six SSR markers (Sat_134, Sat_405, Satt143, Satt398, Sat_195, and Satt694) and 2 amplified fragment length polymorphism markers identified previously were mapped at the same position of the Cgdef gene. These markers should enable to conduct map-based cloning of the Cgdef gene.

Coordinated expression of functionally diverse fructosyltransferase genes is associated with fructan accumulation in response to low temperature in perennial ryegrass.

* Fructan is the major nonstructural carbohydrate reserve in temperate grasses. To understand regulatory mechanisms in fructan synthesis and adaptation to cold environments, the isolation, functional characterization and genetic mapping of fructosyltransferase (FT) genes in perennial ryegrass (Lolium perenne) are described. * Six cDNAs (prft1-prft6) encoding FTs were isolated from cold-treated ryegrass plants, and three were positioned on a perennial ryegrass linkage map. Recombinant proteins were produced in Pichia pastoris and enzymatic activity was characterized. Changes in carbohydrate levels and mRNA levels of FT genes during cold treatment were also analysed. * One gene encodes sucrose-sucrose 1-fructosyltransferase (1-SST), and two gene encode fructan-fructan 6G-fructosyltransferase (6G-FFT). Protein sequences for the other genes (prfts 1, 2 and 6) were similar to sucrose-fructan 6-fructosyltransferase (6-SFT). The 1-SST and prft1 genes were colocalized with an invertase gene on the ryegrass linkage map. The mRNA levels of prft1 and prft2 increased gradually during cold treatment, while those of the 1-SST and 6G-FFT genes first increased, but then decreased before increasing again during a longer period of cold treatment. * Thus at least two different patterns of gene expression have developed during the evolution of functionally diverse FT genes, which are associated in a coordinated way with fructan synthesis in a cold environment.

Spatial and temporal control of transcription of the soybean beta-conglycinin alpha subunit gene is conferred by its proximal promoter region and accounts for the unequal distribution of the protein during embryogenesis.

Differentiation into specific embryo cell types correlates with the processes that lead to the accumulation of seed storage proteins in plants. The alpha subunit of beta-conglycinin, a major component of seed storage proteins in soybean, accumulates at a higher level in cotyledons than in the embryonic axis in developing embryos. To understand the mechanisms underlying this phenomenon, we characterized the upstream region of the alpha subunit gene in terms of transcriptional control using transgenic Arabidopsis thaliana plants carrying reporter gene constructs comprising the 1357-bp upstream sequence of the alpha subunit gene and the beta-glucuronidase (GUS) gene. Analysis of the time-course-dependent pattern of GUS expression revealed that the expression was first confined to the cotyledons and occurred later in the entire embryo during embryogenesis. The level of GUS expression was higher in cotyledons than in the embryonic axis throughout the period of its expression, coincident with the distribution of the alpha subunit protein in soybean embryos. By testing progressively shorter promoter fragments, the cis-acting elements responsible for transcriptional activation in the cotyledons and the embryonic axis were both localized to the region spanning -245 to -161 relative to the transcription start site. It is also concluded that the upstream region up to -245 is sufficient to control the spatial and temporal pattern of transcription, while further upstream regions influence transcription rate without affecting the transcriptional pattern. Overall, these results indicate that the unequal distribution of alpha subunit protein within the embryos is established primarily as a consequence of differential transcriptional activation controlled by a short proximal promoter region of the gene in different embryonic tissues.

Functional analysis of soybean genes involved in flavonoid biosynthesis by virus-induced gene silencing.

Virus-induced gene silencing (VIGS) is a powerful tool for functional analysis of genes in plants. A wide-host-range VIGS vector, which was developed based on the Cucumber mosaic virus (CMV), was tested for its ability to silence endogenous genes involved in flavonoid biosynthesis in soybean. Symptomless infection was established using a pseudorecombinant virus, which enabled detection of specific changes in metabolite content by VIGS. It has been demonstrated that the yellow seed coat phenotype of various cultivated soybean lines that lack anthocyanin pigmentation is induced by natural degradation of chalcone synthase (CHS) mRNA. When soybean plants with brown seed coats were infected with a virus that contains the CHS gene sequence, the colour of the seed coats changed to yellow, which indicates that the naturally occurring RNA silencing is reproduced by VIGS. In addition, CHS VIGS consequently led to a decrease in isoflavone content in seeds. VIGS was also tested on the putative flavonoid 3'-hydroxylase (F3'H) gene in the pathway. This experiment resulted in a decrease in the content of quercetin relative to kaempferol in the upper leaves after viral infection, which suggests that the putative gene actually encodes the F3'H protein. In both experiments, a marked decrease in the target mRNA and accumulation of short interfering RNAs were detected, indicating that sequence-specific mRNA degradation was induced. The present report is a successful demonstration of the application of VIGS for genes involved in flavonoid biosynthesis in plants; the CMV-based VIGS system provides an efficient tool for functional analysis of soybean genes.

[Derivative spectrophotometric and NMR spectroscopic study in pharmaceutical science].

This review starts with an introduction of derivative spectrophotometry followed by a description on the construction of a personal computer-assisted derivative spectrophotometric (DS) system. An acquisition system for inputting digitalized absorption spectra into personal computers and a BASIC program for calculating derivative spectra were developed. Then, applications of the system to drug analyses that are difficult with traditional absorption methods are described. Following this, studies on the interactions of drugs with biological macromolecules by the DS and NMR methods were discussed. An (1)H NMR study elucidated that the small unilamellar vesicle (SUV) has a single membrane made of a phosphatidylcholine bilayer, and that chlorpromazine interacts with both the outer and inner layers. (13)C NMR revealed a reduction of the dissociation constants of phenothiazine drugs due to their interaction with SUV. The partition coefficients of phenothiazine, benzodiazepine and steroid drugs in an SUV-water system and the effects of cholesterol or amino lipids content on these partition coefficients were examined by the DS method. The binding constants of phenothiazine drugs to bovine serum albumin (BSA) and the influence of Na(+), K(+), Cl(-), Br(-), and I(-) on these binding constants were determined by DS. It was found that I(-), Br(-), Cl(-) reduce the binding constants in this order, and that Na(+) and K(+) have no effect. A (19)F NMR study revealed that triflupromazine binds to BSA and human serum albumin in two regions including Site II with different populations, and that a nonsteroidal anti-inflammatory drug, niflumic acid, binds Sites Ia and Ib.

Isolation and molecular characterization of a Lotus japonicus R2R3-MYB subgroup 7 transcription factor gene.

We isolated an ortholog (LjMYB12) of the Arabidopsis R2R3-MYB transcription factor (TF) gene from Lotus japonicus to investigate the regulation of flavonoid biosynthesis, which is driven by many paralogous genes in L. japonicus. We characterized the spatial and temporal expression of LjMYB12 in leaves, stems, roots, flowers, immature seeds, seedling leaves, and seedling roots. Expression was much higher in flowers than in other tissues. To verify the relationship between the expression of LjMYB12 and that of flavonoid biosynthesis genes, we generated transgenic L. japonicus plants overexpressing LjMYB12. Overexpression of LjMYB12 resulted in the upregulation of genes for a chalcone synthase paralog (CHS1), flavanone 3-hydroxylase, and flavonol synthase. Interestingly, LjMYB12 strongly activated CHS1 but did not activate other CHS paralogs. This result suggests differences in the spatial or temporal activation of CHS paralogs by R2R3-MYB TFs. Molecular characterization of R2R3-MYB TFs in L. japonicus will reveal the effects of gene duplication on the regulation of diverse flavonoid biosynthesis.

Dissociation constants of phenothiazine drugs incorporated in phosphatidylcholine bilayer of small unilamellar vesicles as determined by carbon-13 nuclear magnetic resonance spectrometric titration.

The dissociation constants (pKms) of the phenothiazine drugs promazine, chlorpromazine, and triflupromazine, incorporated in the phosphatidylcholine (PC) bilayer of small unilamellar vesicles (SUV), were investigated by a 13C nuclear magnetic resonance (NMR) titration method employing their N-13CH3 (ionizable group) labelled derivatives. Use of the labelled drugs enabled direct observations of the ionization equilibrium of the N-dimethyl group. A second derivative spectrophotometric study proved that 95-98% of the phenothiazine species in the sample solutions (200 microM phenothiazine in the presence of 27 mM PC SUV) were incorporated into the PC bilayer, which simplified the calculation of pKm values by allowing that the phenothiazines in the aqueous phase could be neglected. The pKm values were calculated from the chemical shift dependence of the N-dimethyl 13C NMR signal on the pH value of sample solutions. The pKm values obtained were smaller than those measured in aqueous solutions by about one unit. The existence of cholesterol (30 mol%) in the PC bilayer showed little effect on the pKm values, suggesting that cholesterol in the bilayer does not largely affect the interfacial region where the N-dimethyl group of the incorporated phenothiazines is located. The results offered clear evidence for the pKm decrease and provided their precise values.

Needle-type ultra micro silver/silver chloride reference electrode for use in micro-electrochemistry.

A needle-type ultra micro silver/silver chloride reference electrode having a micro capillary with outer and inner diameters of 1.0 microm and 0.5 +/- 0.2 microm, respectively, was constructed. This micro reference electrode can be stuck into a living cell, and is applicable to use in very small environments, such as an electrochemical cell of an electrochemical scanning tunneling microscope or the detection portion of a micro-TAS. Excellent stability and repeatability of the micro reference electrode potential could be obtained by filling the micro capillary with agar gel containing 3.33 mol/L potassium chloride as a salt bridge, by covering the bare part of the silver wire on which silver chloride was deposited, and by electromagnetic shielding of the measurement cell and wire lead from the electromagnetic waves. The electrode showed stable potential for 7 days after its fabrication using 3.3 mol/L potassium chloride aqueous solution containing silver chloride as an internal electrolyte solution. The electrode exhibited constant electrode potential and excellent stability in test solutions of pH 5-9. The electrode potential of a commercial pH glass electrode measured against the micro reference electrode in standard pH buffer solutions was in good accordance with the Nernst equation.

[Simple method for quantitative determination of active polymorphous and amorphous drugs in drug products by fourier transform-Raman spectroscopy].

The feasibility of a simple Fourier transform (FT)-Raman spectroscopic method for the quantitative determination of unexpected active drug polymorphs or amorphous in drug products was explored. In this study, calibration samples were prepared by physically mixing drug substances with their polymorphs or amorphous, without using excipients. A partial least-squares (PLS) method was applied to the quantitative analysis of the FT-Raman spectra obtained. As model drug products, compound A drug substances (form alpha) containing several ratios of polymorph, (form beta), were physically mixed with excipients to prepare powder samples corresponding to compound A tablets (30 and 120 mg). The mixture of compound B, form I and amorphous, were also mixed with excipients to mimic powdered compound B tablets (32 mg). Satisfactory relationships between the theoretical contents of polymorph or amorphous and determined contents were obtained; quantitation limits were 5-10%. In the case of powder samples corresponding to compound A tablets (30 mg) and compound B tablets (32 mg), differentiating the FT-Raman spectra prior to PLS analysis was required to improve the precision of the determinations.

Partitioning of organophosphorus pesticides into phosphatidylcholine small unilamellar vesicles studied by second-derivative spectrophotometry.

In order to quantitatively examine the lipophilicity of the widely used organophosphorus pesticides (OPs) chlorfenvinphos (CFVP), chlorpyrifos-methyl (CPFM), diazinon (DZN), fenitrothion (FNT), fenthion (FT), isofenphos (IFP), profenofos (PFF) and pyraclofos (PCF), their partition coefficient (Kp) values between phosphatidylcholine (PC) small unilamellar vesicles (SUVs) and water (liposome-water system) were determined by second-derivative spectrophotometry. The second-derivative spectra of these OPs in the presence of PC SUV showed a bathochromic shift according to the increase in PC concentration and distinct derivative isosbestic points, demonstrating the complete elimination of the residual background signal effects that were observed in the absorption spectra. The Kp values were calculated from the second-derivative intensity change induced by addition of PC SUV and obtained with a good precision of R.S.D. below 10%. The Kp values were in the order of CPFM>FT>PFF>PCF>IFP>CFVP>FNT⩾DZN and did not show a linear correlation relationship with the reported partition coefficients obtained using an n-octanol-water system (R(2)=0.530). Also, the results quantitatively clarified the effect of chemical-group substitution in OPs on their lipophilicity. Since the partition coefficient for the liposome-water system is more effective for modeling the quantitative structure-activity relationship than that for the n-octanol-water system, the obtained results are toxicologically important for estimating the accumulation of these OPs in human cell membranes.

Partition of N-monodemethylated phenothiazine drugs to phosphatidylcholine bilayer vesicles studied by second-derivative spectrophotometry.

The partition coefficients (Kps) of three N-monodemethylated metabolites of phenothiazines (chlorpromazine (CPZ), triflupromazine, and promazine) between phosphatidylcholine (PC) vesicles (SUV) and water were determined to evaluate their affinity to biomembranes by second-derivative spectrophotometry without any separation procedures. The second derivative spectra showed distinct derivative isosbestic points confirming the entire elimination of the residual background signal effects of the SUV, which were observed in the absorption spectra. From the relationship between PC SUV concentration and the derivative intensity change (DeltaD) of each metabolite induced by its interaction with the PC bilayer, the Kp values were calculated and could be obtained with the R.S.D. of below 10% (n = 5) proving an accuracy of the derivative method. The obtained Kp values were similar to those of the parent drugs (the relative differences were within 15%), although the partition coefficient of N-monodemethylated CPZ measured in an octanol/buffer system was reported to be about 1/18 of CPZ. The results obtained from the PC liposome/buffer system do not contradict with psychoactivity and brain accumulation of N-monodemethylated metabolites of phenothiazines, in contrast to the results of the octanol/buffer system.

19F NMR spectroscopic study on the binding of triflupromazine to bovine and human serum albumins.

The 19F NMR spectrum of triflupromazine hydrochloride (TFZ) in a buffer solution (pH 6.8) showed a single sharp signal of the TFZ CF3 group at 13.5 ppm from the external trifluoroacetic acid. The addition of 1 mM HSA or BSA to the sample solution caused a split of the CF3 signal into two broadened signals shifted to slightly lower (0.2 ppm) and higher (0.7 ppm) fields, respectively, from the original position. Denaturation of the albumins by guanidine hydrochloride (3M) restored the two broadened signals to a slightly broadened single signal, indicating that TFZ has at least two binding sites on HSA and BSA, respectively. From the competitive binding 19F NMR experiments using Warfarin (Site-I ligand), l-tryptophan (Site-II ligand), NaCl, and oleate, the signal at high field was assigned to the TFZ bound to Site II. Comparison of the signal intensity revealed that the affinity of TFZ for Site II on HSA was considerably higher than that on BSA. The low-field signal could be identified as a weight-averaged signal between nonspecifically bound TFZ to HSA (BSA) and free TFZ in the water phase. In the presence of physiological concentrations of NaCl, major binding of TFZ to HSA and BSA was considered to be nonspecific. The present work indicates that 19F NMR is very useful for obtaining important detailed information regarding the binding of fluorinated drugs to serum albumins.