The metabolism of 7,10,13,16,19-docosapentaenoic acid to 4,7,10,13,16,19-docosahexaenoic acid in rat liver is independent of a 4-desaturase.

The hypothesis that the last step in the biosynthesis of 4,7,10,13,16,19-22:6 from linolenate is catalyzed by an acyl-CoA-dependent 4-desaturase has never been evaluated by direct experimentation. When rat liver microsomes were incubated with [1-14C]7,10,13,16,19-22:5, under conditions where linoleate was readily desaturated to 6,9,12-18:3, it was never possible to detect the product of the putative 4-desaturase. In the presence of malonyl-CoA, 7,10,13,16,19-22:5 was sequentially chain-elongated to 9,12,15,18,21-24:5, followed by its desaturation at position 6 to give 6,9,12,15,18,21-24:6. Microsomes desaturated 9,12,15,18,21-24:5 at rates similar to those observed for metabolizing linoleate to 6,9,12-18:3. Rat hepatocytes metabolize [1-14C]7,10,13,16,19-22:5 to 22:6(n-3), but in addition, it was possible to detect small amounts of esterified 24:5(n-3) and 24:6(n-3) in phospholipids, which is a finding consistent with their role as obligatory intermediates in 22:6(n-3) biosynthesis. When 3-14C-labeled 24:5(n-3) or 24:6(n-3) were incubated with hepatocytes, only a small amount of either substrate was esterified. [3-14C] 24:5(n-3) was metabolized both by beta-oxidation to 22:5(n-3) and by serving as a precursor for the biosynthesis of 24:6(n-3) and 22:6(n-3). The primary metabolic fate of [3-14C]24:6(n-3) was beta-oxidation to 22:6(n-3), followed by its acylation into membrane lipids. Our results thus document that 22:5(n-3) is the precursor for 22:6(n-3) but via a pathway that is independent of a 4-desaturase. This pathway involves the microsomal chain elongation of 22:5(n-3) to 24:5(n-3), followed by its desaturation to 24:6(n-3). This microsomal product is then metabolized, via beta-oxidation, to 22:6(n-3).     

Morphological and Functional Association of Sec22b/ERS-24 with the pre-Golgi Intermediate Compartment

Yeast Sec22p participates in both anterograde and retrograde vesicular transport between the endoplasmic reticulum (ER) and the Golgi apparatus by functioning as a v-SNARE (soluble N-ethylmaleimide-sensitive factor [NSF] attachment protein receptor) of transport vesicles. Three mammalian proteins homologous to Sec22p have been identified and are referred to as Sec22a, Sec22b/ERS-24, and Sec22c, respectively. The existence of three homologous proteins in mammalian cells calls for detailed cell biological and functional examinations of each individual protein. The epitope-tagged forms of all three proteins have been shown to be primarily associated with the ER, although functional examination has not been carefully performed for any one of them. In this study, using antibodies specific for Sec22b/ERS-24, it is revealed that endogenous Sec22b/ERS-24 is associated with vesicular structures in both the perinuclear Golgi and peripheral regions. Colabeling experiments for Sec22b/ERS-24 with Golgi mannosidase II, the KDEL receptor, and the envelope glycoprotein G (VSVG) of vesicular stomatitis virus (VSV) en route from the ER to the Golgi under normal, brefeldin A, or nocodazole-treated cells suggest that Sec22b/ERS-24 is enriched in the pre-Golgi intermediate compartment (IC). In a well-established semi-intact cell system that reconstitutes transport from the ER to the Golgi, transport of VSVG is inhibited by antibodies against Sec22b/ERS-24. EGTA is known to inhibit ER–Golgi transport at a stage after vesicle/transport intermediate docking but before the actual fusion event. Antibodies against Sec22b/ERS-24 inhibit ER–Golgi transport only when they are added before the EGTA-sensitive stage. Transport of VSVG accumulated in pre-Golgi IC by incubation at 15°C is also inhibited by Sec22b/ERS-24 antibodies. Morphologically, VSVG is transported from the ER to the Golgi apparatus via vesicular intermediates that scatter in the peripheral as well as the Golgi regions. In the presence of antibodies against Sec22b/ERS-24, VSVG is seen to accumulate in these intermediates, suggesting that Sec22b/ERS-24 functions at the level of the IC in ER–Golgi transport.     

Five new polar sterols from the black coral Antipathes subpinnata.

In addition to the known (20S,22E)-cholesta-1,4,22-triene-16 beta,18, 20-triol-3-one, (20S,22E)-24-methyl-cholesta-1,4,22-triene-16 beta,18,20-triol-3-one, and (20S,22E)-24-methylcholesta-4,22-diene-16 beta,18,20-triol-3-one, the methanol extract of the Mediterranean anthozoan Antipathes subpinnata was shown to contain five new sterols: (20S,22E)-cholesta-1,4,22-triene-18,20-diol-3-one, (20S,22E)-24-methylcholesta-1,4,22-triene-18,20-diol-3-one, (20S)-cholest-4-ene-16 beta,18,20-triol-3-one, (20S,22E)-cholesta-4,22-diene- 16 beta,18,20-triol-3-one, and (20S,22E)-24-methylcholesta-4,22-diene-16 beta,18,20-triol-3-one, whose stereostructures were elucidated on the basis of physicochemical evidence. The previously unassigned chirality at C-20 of the known sterols has been also established as S.     

A novel sterol from Chinese truffles Tuber indicum

From the fruiting bodies of Ascomycetes Tuber indicum, a new steroidal glucoside with polyhydroxy ergosterol nucleus, tuberoside (2), has been isolated along with additional four known ergosterol derivatives, (22E, 24R)-ergosta-7, 22-dien-3beta, 5alpha, 6beta-triol (1), 5alpha, 8alpha-epidioxy-(22E, 24R)-ergosta-6, 22-dien-3beta-ol (3), (22E, 24R)-ergosta-5, 22-dien-3beta-ol (4), and (22E, 24R)-ergosta-4, 6, 8(14), 22-tetraen-3-one (5). The structure of new compound was established as 3-O-beta-D-glucopyranosyl-(22E, 24R)-ergosta-7, 22-dien-5alpha, 6beta-diol (2) on the basis of chemical and spectroscopic means ((1)H NMR, (13)C NMR, HMQC, HMBC, MS, and IR). This is the first example of isolation of a polyhydroxylated ergosterol glucoside from higher fungi in nature.     

Steroids in Porifera. II. Steroid derivatives from two sponges of the family Halichondriidae. Sokotrasterol sulfate, a marine steroid with a new pattern of side chain alkylation

A trisulfated derivative of 24,25,26,26-tetramethyl-5 alpha-cholest-23E-ene-2 alpha, 3 beta, 6 alpha-triol (sokotrasterol sulfate) has been isolated from the sponge Halichondriidae gen. sp., collected near Sokotra Island (Arabian Sea), and its structure has been elucidated. The side chain of the new steroid involves a "normal" alkylation at C-24 and the unprecedented addition of two extra methyl groups at C-26 and one extra methyl group at C-25. A free sterol fraction contained only 24-isopropyl-5-cholesten-3 beta-ol and 24-isopropyl-5, 22E-cholestadien-3 beta-ol. 24-Isopropyl-5, 22E-cholestadien-3 beta-ol as sole monohydroxy sterol and halistanol sulfate as major polyhydroxylated steroid derivative have been detected in Halichondria sp., a Madagascar sponge.     

Ecogeographic variability and differentiation of Japanese dwarf pine populations in Kamchatka Krai with respect to the characters of generative organs

The assessment of ecogeographic variability and differentiation of 22 cenopopulations of Japanese dwarf pine Pinus pumila (Pall.) Regel (Pinaceae) on the Kamchatka Peninsula and in Koryakia has been carried out in regard to morphological and phenotypic characters of mature seed cones. The viability of seeds in the cones of ten populations has been determined by a radiographic method. A latitudinal nature of phenotypic differentiation of cenopopulations in the region has been revealed. Lower seed viability related to a high percent of seeds without embryos or with underdeveloped embryos has been recorded in the northeastern populations. The average share of viable seeds in the region is 52% and in the northeastern populations it reaches only 6–24%.     

Six new ergosterols from the marine-derived fungus Rhizopus sp

Six new ergosterols, including 3beta-hydroxyl-(22E, 24R)-ergosta-5,8,22-trien-7,15-dione (1), 3beta-hydroxyl-(22E, 24R)-ergosta-5,8,14,22-tetraen-7-one (2), 3beta,15beta-dihydroxyl-(22E, 24R)-ergosta-5,8(14),22-trien-7-one (3), 3beta,15alpha-dihydroxyl-(22E, 24R)-ergosta-5,8(14),22-trien-7-one (4), 3beta-hydroxyl-(22E, 24R)-ergosta-5,8(14),22-trien-7,15-dione (5), and 5alpha,8alpha-epidioxy-23,24(R)-dimethylcholesta-6,9(11),22-trien-3beta-ol (6), have been isolated from the marine-derived fungus Rhizopus sp., along with four known ones (7-10). The structures of the new compounds were determined on the basis of extensive spectroscopic data. All compounds were evaluated for their cytotoxic activities on P388, A549, HL-60, and BEL-7420 cell lines by the MTT and SRB methods.     

New polyoxygenated steroids from the Antarctic octocoral Dasystenella acanthina

The chemical study of the Antarctic octocoral Dasystenella acanthina has led to the isolation of the new polyoxygenated steroids (24R,22E)-24-hydroxycholest-4,22-dien-3-one (1), 23-acetoxy-24,25-epoxycholest-4-en-3-one (2), 12beta-acetoxycholest-4-en-3,24-dione (3), 12beta-acetoxy-24,25-epoxycholest-4-en-3-one (4), (22E)-25-hydroxy-24-norcholest-4,22-dien-3-one (5), 3alpha-acetoxy-25-hydroxycholest-4-en-6-one (6), and 3alpha,11alpha-diacetoxy-25-hydroxycholest-4-en-6-one (7), whose structures have been established by spectroscopic analysis. The absolute stereochemistry at C-24 in compound 1 has been determined through the 1H NMR study of the corresponding (R)- and (S)-MPA esters. All the new compounds showed significant activities as growth inhibitors of several human tumor cell lines. In addition, cytostatic and cytotoxic effects were also observed on selected tumor cell lines.     

PCR-assisted localization of the human SRPR gene

Summary A polymerase chain reaction (PCR)-based assay has been designed that detects the presence of the human signal recognition particle receptor (SRPR) gene in inter-species somatic cell hybrids. By using hybrids containing various fragments of chromosome 11q, SRPR has been mapped to a chromosomal region flanked by the 11q23 and 11q24 breakpoints associated with the constitutional and neuroepithelioma (11;22) translocations, respectively.     

Interactions of phospholipids with the potassium channel KcsA

The potassium channel KcsA from Streptomyces lividans has been reconstituted into bilayers of phosphatidylcholines and fluorescence spectroscopy has been used to characterize the response of KcsA to changes in bilayer thickness. The Trp residues in KcsA form two bands, one on each side of the membrane. Trp fluorescence emission spectra and the proportion of the Trp fluorescence intensity quenchable by I(-) hardly vary in the lipid chain length range C10 to C24, suggesting efficient hydrophobic matching between KcsA and the lipid bilayer over this range. Measurements of fluorescence quenching for KcsA reconstituted into mixtures of brominated and nonbrominated phospholipids have been analyzed to give binding constants of lipids for KcsA, relative to that for dioleoylphosphatidylcholine (di(C18:1)PC). Relative lipid binding constants increase by only a factor of three with increasing chain length from C10 to C22 with a decrease from C22 to C24. Strongest binding to di(C22:1)PC corresponds to a state in which the side chains of the lipid-exposed Trp residues are likely to be located within the hydrocarbon core of the lipid bilayer. It is suggested that matching of KcsA to thinner bilayers than di(C24:1)PC is achieved by tilting of the transmembrane alpha-helices in KcsA. Measurements of fluorescence quenching of KcsA in bilayers of brominated phospholipids as a function of phospholipid chain length suggest that in the chain length range C14 to C18 the Trp residues move further away from the center of the lipid bilayer with increasing chain length, which can be partly explained by a decrease in helix tilt angle with increasing bilayer thickness. In the chain length range C18 to C24 it is suggested that the Trp residues become more buried within the hydrocarbon core of the bilayer.     

Hippocampal lipids linked to spatial memory in the C57bl/6j mouse

Although the role of individual brain lipids for learning and memory has been reported, no systematic approach associating brain lipids with spatial memory has been carried out. It was therefore the aim of the study to determine brain lipids in hippocampus of mice forming and yoked controls that did not form spatial memory using the probe trial as the endpoint.10 animals were trained in the Morris water maze (MWM) and 10 mice were serving as yoked controls i.e. no platform was used during the whole experiment.Hippocampal tissue lipids were extracted and data were acquired with Fourier transformation ion cyclotron resonance mass spectrometry (LTQ-FT) coupled to HPLC.Glycerophosphatidylethanolamines (18:0/22:6, 18:0/20:4 and 18:1/18:1), plasmalogens (16:0-10/22:6 and 18:0-10/22:6) and ceramides (18:0) showed higher levels in the trained group, while glycerolysophosphatidylcholines (16:0, 18:1, 18:0, 20:4), sphingomyelins (16:0, 24:1), ether linked glycerophosphatidylcholines (16:0-10/18:0), glycerophosphatidylcholines (16:0/18:1, 16:0/18:0, 18:0/18:1, 38:7, 18:1/20:1, 20:4/20:4, 22:1/18:1, 22:0/18:1, 20:4/22:6, 22:6/22:6), glucosylceramide (24:1) and plasmalogen (18:0-10/20:1) revealed lower levels in the trained group.Decreased levels of certain species of lysophosphatidylcholine, sphingomyelin, plasmenylphosphatidylcholine, phosphatidylcholine, glycosylceramide and plasmalogen at the probe trial for spatial memory may indicate catabolism in terms of consumption during this process.Increased hippocampal levels of long chain highly unsaturated phosphatidylethanolamines, plasmalogens and ceramides may reflect increased synthesis or decreased degradation at the endpoint of memory testing, probably representing interactions in the brain lipid pathways. The study shows pathways involved in spatial memory, may propose the use of individual brain lipids as probable cognitive enhancers and forms the basis for further studies on the role of brain lipids per se.     

Age specificity of lipid fluidity in the sarcoplasmic reticulum of the rat heart

The lipid fluidity in heart sarcoplasmic reticulum membranes prepared from adult (12 mo.) and old (24 mo.) rats has been measured by the fluorescence probe (DPPH) and spin probe (5NS) methods at 22 and 37 degrees C. The lipid fluidity in the old rat membranes is higher than that in the adult rat ones. It has been suggested that this difference is caused by age lowering in reliability of membrane fluidity stabilization systems.     

Hippocampal lipids linked to spatial memory in the C57BL/6J mouse

Although the role of individual brain lipids for learning and memory has been reported, no systematic approach associating brain lipids with spatial memory has been carried out. It was therefore the aim of the study to determine brain lipids in hippocampus of mice forming and yoked controls that did not form spatial memory using the probe trial as the endpoint. 10 animals were trained in the Morris water maze (MWM) and 10 mice were serving as yoked controls i.e. no platform was used during the whole experiment. Hippocampal tissue lipids were extracted and data were acquired with Fourier transformation ion cyclotron resonance mass spectrometry (LTQ-FT) coupled to HPLC. Glycerophosphatidylethanolamines (18:0/22:6, 18:0/20:4 and 18:1/18:1), plasmalogens (16:0-10/22:6 and 18:0-10/22:6) and ceramides (18:0) showed higher levels in the trained group, while glycerolysophosphatidylcholines (16:0, 18:1, 18:0, 20:4), sphingomyelins (16:0, 24:1), ether linked glycerophosphatidylcholines (16:0-10/18:0), glycerophosphatidylcholines (16:0/18:1, 16:0/18:0, 18:0/18:1, 38:7, 18:1/20:1, 20:4/20:4, 22:1/18:1, 22:0/18:1, 20:4/22:6, 22:6/22:6), glucosylceramide (24:1) and plasmalogen (18:0-10/20:1) revealed lower levels in the trained group. Decreased levels of certain species of lysophosphatidylcholine, sphingomyelin, plasmenylphosphatidylcholine, phosphatidylcholine, glycosylceramide and plasmalogen at the probe trial for spatial memory may indicate catabolism in terms of consumption during this process. Increased hippocampal levels of long chain highly unsaturated phosphatidylethanolamines, plasmalogens and ceramides may reflect increased synthesis or decreased degradation at the endpoint of memory testing, probably representing interactions in the brain lipid pathways. The study shows pathways involved in spatial memory, may propose the use of individual brain lipids as probable cognitive enhancers and forms the basis for further studies on the role of brain lipids per se.     

Three new polyhydroxysteroids from the tropical starfish Asteropsis carinifera

Thirteen steroidal compounds including three new polyhydroxysteroids, (24R,25S)-24-methyl-5α-cholestane-3β,6α,8,15β,16β,26-hexaol, (22E,24R,25S)-24-methyl-5α-cholest-22-ene-3β,6α,8,15β,16β,26-hexaol and (22E,24R,25S)-24-methyl-5α-cholest-22-ene-3β,4β,6α,8,15β,16β,26-heptaol, have been isolated along with the previously known ten polyhydroxysteroids from the tropical starfish Asteropsis carinifera collected near the coast of Vietnam. The structures of new compounds were elucidated by spectroscopic methods (mainly 2D NMR and ESI-mass-spectrometry).     

Maturational changes in T4 to T3 conversion in domestic fowl

The in vivo conversion of thyroxine (T4) to triiodothyronine (T3) has been determined in fed and 24 hr-fasted thyroidectomized cockerels at 4, 7, 13 and 23 weeks of age. The conversion of T4 to T3 in pubertal (13-week-old) and adult (22-week-old) cockerels was greater than that in immature (less than 7-week-old) chicks. The deprivation of food for 24 hr markedly reduced the rate of T4 to T3 conversion, especially in immature chicks. These maturational changes in T4 to T3 conversion may be related to differences in metabolic rate.     

Preparation and characterization of 3-monohydroxylated bile acids of different side chain length and configuration at C-3. Novel approach to the synthesis of 24-norlithocholic acid

A series of 3-monohydroxylated bile acids, in unlabeled and radioactive form, of varying side chain length and configuration at C-3 has been synthesized and rigorously characterized. They include: 3 alpha- and 3 beta-hydroxy-5 beta-androstane-17 beta-carboxylic acids (C20); 3 alpha- and 3 beta-hydroxy-5 beta-pregnan-21-oic acids (C21); 3 alpha- and 3 beta-hydroxy-23,24-bisnor-5 beta-cholan-22-oic acids (C22); 3 alpha- and 3 beta-hydroxy-24-nor-5 beta-cholan-23-oic acids (C23, norlithocholic and isonorlithocholic acids); and 3 beta-hydroxy-5 beta-cholan-24-oic acid (C24, isolithocholic acid). A novel approach to the degradation of lithocholic acid acetate to 24-norlithocholic acid is described. This degradation involves the photochemical modification of a Hunsdiecker reaction and Kornblum oxidation of the intermediate 23-bromide. The availability of these compounds makes it possible to study the metabolism and biological effects of short chain bile acids.     

H2O2 production downstream of FLT3 is mediated by p22phox in the endoplasmic reticulum and is required for STAT5 signalling

The internal tandem duplication (ITD) of the juxtamembrane region of the FLT3 receptor has been associated with increased reactive oxygen species (ROS) generation in acute myeloid leukemia (AML). How this elevated level of ROS contributes to the leukemic phenotype, however, remains poorly understood. In this work we show that ROS in the FLT3-ITD expressing AML cell line MV4-11 is reduced by treatment with PKC412, an inhibitor of FLT3, DPI, a flavoprotein inhibitor, and VAS2870, a Nox specific inhibitor, suggesting that ROS production is both FLT3 and NADPH oxidase dependent. The majority of these ROS co-localize to the endoplasmic reticulum (ER), as determined with the H(2)O(2)-specific aryl-boronate dye Peroxyorange 1, which also corresponds to co-localization of p22phox. Moreover, knocking down p22phox dramatically reduces H(2)O(2) after 24 hours in the ER, without affecting mitochondrial ROS. Significantly, the FLT3 inhibitor PKC412 reduces H(2)O(2) in FLT3-ITD expressing cell lines (MV4-11, MOLM-13) through reduction of p22phox over 24 hours. Reduced p22phox is achieved by proteasomal degradation and is prevented upon GSK3-β inhibition. Knockdown of p22phox resulted in reduced STAT5 signalling and reduced Pim-1 levels in the cells after 24 hours. Thus, we have shown that FLT3 driven H(2)O(2) production in AML cells is mediated by p22phox and is critical for STAT5 signalling.     

Hedgehog signaling: from the Drosophila cuticle to anti-cancer drugs

Hh signaling controls cell proliferation and differentiation in processes that range from insect segmentation and limb formation to vertebrate neural tube development and bone differentiation. Moreover, Hh signaling appears to regulate stem cell homeostasis in adult tissues, while persistent Hh pathway activity has pathological consequences in a number of cancers. Two recent meetings, a Karolinska Institute Nobel conference (August 22-24, 2004) and a joint EMBO and Juan March Institute workshop (October 25-27, 2004), provided the opportunity to take stock of the progress that has been made in understanding Hh signaling and also to remind us of the many questions that still remain unanswered.     

Subcellular distribution of the different platelet proteins phosphorylated on exposure of intact platelets to ionophore A23187 or to prostaglandin E1. Possible role of a membrane phosphopolypeptide in the regulation of calcium-ion transport.

Exposure of 32P-labelled human platelets to ionophore A23187 results in an increased incorporation of 32P into polypeptides with apparent mol.wts. of 47 000 (P47) and 20 000 (P20), whereas exposure to prostaglandin E1 results in increased labelling of polypeptides with apparent mol.wts. of 24 000 (P24) and 22 000 (P22) [Haslam, Lynham & Fox (1979) Biochem. J. 178, 397-406]. Labelled platelets that had been incubated with ionophore A23187 or prostaglandin E1 were sonicated and rapidly separated into three fractions by differential centrifugation. Electron microscopy and measurement of marker enzymes indicated that the 1300-19 000 gav. particulate fraction was enriched in granules, mitochondria and plasma membranes, that the 19 000-90 000 gav. particulate fraction was enriched in both intracellular and plasma membranes and that the 90 000 gav. supernatant contained only soluble proteins. 32P-labelled phosphopolypeptide P47 was present almost exclusively in the 90 000 gav. supernatant, whereas phosphopolypeptide P20 was largely dephosphorylated under fractionation conditions that protected other phosphopolypeptides. 32P-labelled phosphopolypeptide P24 was enriched in both particulate fractions, but particularly in the 19 000-90 000 gav. fraction, and may therefore be present in both the intracellular and plasma membranes. Phosphopolypeptide P22 appeared to be similarly distributed. Both particulate fractions were capable of the ATP-dependent oxalate-stimulated uptake of Ca2+. When the 19 000-90 000 gav. membrane fraction was prepared from platelets that had been incubated with ionophore A23187, active uptake of Ca2+ did not occur, but when this fraction was isolated from platelets that had been exposed to prostaglandin E1, uptake of Ca2+ was significantly greater than observed with the corresponding membranes from control platelets. It is suggested that phosphorylation of polypeptide P24 (or P22) by a cyclic AMP-dependent protein kinase may promote the active transport of Ca2+ out of the platelet cytosol.     

A dwarf mutant strain of Pharbitis nil, Uzukobito (kobito), has defective brassinosteroid biosynthesis

Japanese morning glory (Pharbitis nil) is a model plant characterized by a large stock of spontaneous mutants. The recessive mutant Uzukobito shows strong dwarfism with dark-green rugose leaves. The phenotype was rescued by the application of brassinolide, a bioactive brassinosteroid (BR), indicating that Uzukobito was a BR-deficient mutant. A detailed analysis of the endogenous BR levels in Uzukobito and its parental wild-type plant showed that Uzukobito had a lower level of BRs downstream of (24R)-24-methyl-5alpha-cholestan-3-one and (22S, 24R)-22-hydroxy-24-methyl-5alpha-cholestan-3-one than those in wild-type plants, while their immediate precursors (24R)-24-methylcholest-4-en-3-one and (22S, 24R)-22-hydroxy-24-methylcholest-4-en-3-one accumulated relatively more in Uzukobito. These results indicate that Uzukobito had a defect in the conversion of (24R)-24-methylcholest-4-en-3-one and (22S, 24R)-22-hydroxy-24-methylcholest-4-en-3-one to their 5alpha-reduced forms, which is catalyzed by de-etiolated2 (DET2) in Arabidopsis. The P. nil ortholog of the DET2 gene (PnDET2) was cloned and shown to have the greatest similarity to DET2 among all the putative genes in Arabidopsis. Uzukobito had one amino acid substitution from Glu62 to Val62 in the deduced amino acid sequence of PnDET2. Recombinant PnDET2 expressed in COS-7 cells was found to be a functional steroid 5alpha-reductase (S5alphaR) converting (24R)-24-methylcholest-4-en-3-one to (24R)-24-methyl-5alpha-cholestan-3-one, while PnDET2 with the mutation did not show any catalytic activity. This shows that a plant S5alphaR can convert an intrinsic substrate. All these results clearly demonstrate that the Uzukobito phenotype resulted from a mutation on PnDET2, and a morphological mutant has been characterized at the molecular level among a large stock of P. nil mutants.