Hematopoietic stem cell transplantation for 30 patients with primary immunodeficiency diseases: 20 years experience of a single team.

We retrospectively analyzed our results of 30 patients with three distinctive primary immunodeficiency diseases (PIDs)--severe combined immunodeficiency (SCID, n = 11), Wiskott-Aldrich syndrome (WAS, n = 11) and X-linked hyper-immunoglobulin M (IgM) syndrome (XHIM, n = 8)--who underwent hematopoietic SCT (HSCT) during the past 20 years. Until 1995, all donors were HLA-haploidentical relatives with T-cell depletion (TCD) (n = 8). Since 1996, the donors have been HLA-matched related donors (MRD) (n = 8), unrelated BM (UR-BM) (n = 7) and unrelated cord blood (UR-CB) (n = 7). Twenty-seven of 30 patients had various pre-existing infections with or without organ damages before HSCT. Conditioning regimen and GVHD prophylaxis were determined according to disease, donor and pretransplant status. Although one of eight patients transplanted with TCD is alive with full engraftment, the other seven died. On the other hand, 18 of 22 patients transplanted without TCD are alive and well, including six of eight transplanted from MRD, seven of seven from UR-BM and five of seven from UR-CB. All 19 survivors did not require Ig supplementation after HSCT. These results indicate that UR-CBT as well as UR-BMT provides good results for PID comparable to MRD-SCT, and that early diagnosis, HSCT at early stage, careful supportive therapy and monitoring for various pathogens are important for the successful HSCT.

Pancreaticobiliary maljunction: retrospective and nationwide survey in Japan.

Pancreaticobiliary maljunction (PBM) is a congenital anomaly defined as a union of the pancreatic and biliary duct that is located outside the duodenal wall. The Japanese Study Group on Pancreaticobiliary Maljunction and the Committee for Registration enrolled and analyzed 1627 patients with PBM who had been diagnosed and treated from January 1, 1990 to December 31, 1999 at 141 hospitals throughout the country. There were 1239 patients with dilatation of the bile duct (group A) and 388 patients without dilatation (group B). The average age was 24 years in group A and 47 years in group B; the age was significantly higher in group B. The type of confluence between the terminal choledochus and the pancreatic duct has been classified into three types (type a, right-angle type; type b, acute-angle type; and type c, complex type). In group A, type a accounted for 57.9% and was significantly more frequent compared with the other types (type b, 32.4%; type c, 5.6%). In group B, type b accounted for 60.8%, being significantly more frequent compared with the other types (type a, 29.4%; type c, 7.2%). Subjective symptoms, preoperative complications (e.g., liver dysfunction and acute pancreatitis), pancreatic stone, and pancreatic duct morphological abnormality were significantly more frequent in group A. However, the amylase levels in the bile and gallbladder were significantly higher in group B, and the presence of gallstone and morphological abnormality of the gallbladder was significantly more frequent in group B. The occurrence rate of cancer in the biliary tract was 10.6% in group A and 37.9% in group B, being significantly higher in group B. In group A, cancer of the extrahepatic bile duct was seen in 33.6% and cancer of the gallbladder was seen in 64.9%, but gallbladder cancer was present significantly more frequently in the patients with diffuse or cylindrical dilatation, and bile duct cancer was present significantly more frequently in the patients with cystic dilatation. In group B, 93.2% of the patients had gallbladder cancer, and bile duct cancer was found in as few as 6.8%. Against this background Japanese surgeons regard cholecystectomy, resection of the extrahepatic bile duct, and hepaticojejunostomy as standard operations for PBM with dilatation of the bile duct. However, opinion on whether or not the bile duct should be removed in the treatment of PBM without dilatation of the bile duct has been divided among Japanese surgeons. A randomized controlled trial is necessary.

Sharing of the same embryogenic pathway in anorectal malformations and anterior sacral myelomeningocele formation.

The objective of this study was to determine whether anorectal malformations (ARMs) and anterior sacral myelomeningocele share the same embryogenic pathway in a mouse model. Etretinate (Ro 10-9359) was administrated to C57BL/6 mice on gestation day 9 (E9). Sections of embryos and fetuses from E9.5 to E18 were observed by HE staining. Immunohistochemical staining with anti-NeuN and anti-GFAP was also done to determine cell origins of a presacral mass. In etretinate-treated embryos, neuroepithelial cells proliferated in the presacral region on E9.5. On E12, a canal appeared between the ectopic proliferated neuroepithelium and hindgut. On E13, anorectum abnormally kept a canal with the ventral urogenital tract through a fistula. On E13.5, a huge mass formed in the presacral region. On E18, 76.9% (30/39) of fetuses had ARMs, 100% (39/39) had a presacral mass (71.8% were huge) and 100% (39/39) had a sacral defect. The types of ARMs were mainly rectourethral or rectocloacal fistula. The presacral mass was anterior sacral myelomeningocele. We thus established the first mouse model of the Currarino triad, congenital caudal anomalies, including ARM, sacral abnormality and presacral mass. These disorders share the same embryogenic pathway. The teratogenic target of etretinate is the tail bud. Abnormal differentiation of the tail bud mesenchyme leads to defects of the tailgut and caudal neural tube. The abnormal mass blocks normal descent of the dorsal cloaca through the most posterior part of the cloacal plate.

Outcome of hepatoblastoma treated with the JPLT-1 (Japanese Study Group for Pediatric Liver Tumor) Protocol-1: A report from the Japanese Study Group for Pediatric Liver Tumor.

PURPOSE: Hepatoblastoma is the most common malignant liver tumor in childhood. Multicenter studies elucidate the optimal pre- or postoperative chemotherapeutic regimens. This report reviews the results of the Japanese Study Group for Pediatric Liver Tumor Protocol-1 (JPLT-1) and compares its outcomes with published reports of other studies. METHODS: From March 1991 to December 1999, 154 patients with malignant liver tumor including 145 cases of hepatoblastomas were enrolled in the JPLT study. Data from 134 cases were analyzed in this study. JPLT-1 protocol 91A was used for patients with stage I or II hepatoblastoma. The chemotherapy regimen consisted of repeated courses of cisplatin (CDDP), 40 mg/m(2), and tetrahydropyranyl (THP)-Adriamycin, 30 mg/m(2). JPLT-1 protocol 91B was administered to patients with stage IIIA, IIIB, or IV hepatoblastoma. The chemotherapy regimen consisted of repeated courses of CDDP, 80 mg/m(2), and THP-Adriamycin, 30 mg/m(2)/day for 2 days. Courses were repeated every 4 weeks as tolerated. RESULTS: Seven patients died of chemotherapy-related side effects. Six of them died of sepsis caused by leukopenia and 1 case of liver failure. Overall survival rate (3-year/6-year) was 100%/100% for stage I (n = 9), 100%/95.7% for stage II (n = 32), 76.6%/73.8% for stage IIIA (n = 48), 50.3%/50.3% for stage IIIB (n = 25), 64.8%/38.9% for stage IV (n = 20), and 77.8%/73.4% overall. For stage IIIA and B disease, intravenous chemotherapy was better than intraarterial chemotherapy (66.4% v 38.1% for event-free survival and 69.3% v. 57.1% for overall survival). Patients less than 1 year of age had a better prognosis than older patients, but age was not a significant prognostic factor by multivariate analysis. CONCLUSIONS: The overall and event-free survival rates of the JPLT-1 study of hepatoblastoma were comparable with the results of other multicenter studies in Europe and the United States. The event-free survival rate at 3 years for stage IIIB and IV disease was under 50%. New treatment strategies are needed for patients with advanced hepatoblastoma.

Molecular characterization and chromosomal localization of cytochrome P450 genes involved in the biosynthesis of cyclic hydroxamic acids in hexaploid wheat.

The cyclic hydroxamic acids, 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA) and 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA), are defensive secondary metabolites found in gramineous plants including wheat, maize and rye. cDNAs for five cytochromes P450 (P450s) involved in DIBOA biosynthesis (CYP71C6, CYP71C7v2, CYP71C8v2, CYP71C9v1 and CYP71C9v2) were isolated from seedlings of hexaploid wheat [( Triticum aestivum L. cv. Chinese Spring (2n=6x=42, genomes AABBDD)] by RT-PCR and screening of a cDNA library. CYP71C9v1 and CYP71C9v2 are 97% identical to each other in amino acid and nucleotide sequences. The cloned P450 species showed 76-79% identity at the amino acid level to the corresponding maize P450 species CYP71C1-C4, which are also required for DIBOA biosynthesis. The wheat P450 cDNAs were heterologously expressed in the yeast ( Saccharomyces cerevisiae) strain AH22. Microsome fractions from yeast cells expressing these P450 species catalyzed the same reactions as their maize orthologs. The chromosomes carrying the cyp71C6- C9v1 orthologs were identified by Southern hybridization using aneuploid lines of Chinese Spring wheat. The cyp71C9v1 orthologs were located on the chromosomes of wheat homoeologous group-4. The orthologs of the other P450 genes, cyp71C7v2, cyp71C6 and cyp71C8v2, were located on group-5 chromosomes. The same P450 genes were also present in the three ancestral diploid species of hexaploid wheat, T. monococcum (AA), Aegilops speltoides [BB (approximately SS)] and Ae. squarrosa (DD).

Enhancement of metabolizing herbicides in young tubers of transgenic potato plants with the rat CYP1A1 gene.

A rat P450 monooxygenase gene ( CYP1A1) was introduced into potato plants to enhance the metabolism of the environmental contaminants in subterranean organs. The CYP1A1 gene was kept under the control of the potato patatin promoter to enhance tuber-specific expression. A total of 106 transgenic plants (PAT1A1 plants) were obtained following selection by a resistance test to kanamycin and PCR analysis. PAT1A1 plants treated with 10% exogenous sucrose showed a higher activity of monooxgenase in the leaves than the non-transgenic plants. This indicated that the activity enhanced by 10% sucrose was due to the patatin promoter containing the sucrose-inducted elements. One representative transgenic plant, Ag2197, was selected on the basis of monooxgenase activity in the leaves and Western blot analysis. Ag2197 was found to accumulate a large amount of CYP1A1 mRNA and protein in the developing tuber but not in the mature tuber. The residual herbicides, atrazine and chlortoluron, were analyzed in the micro-tubers of Ag2197 and non-transgenic plants. The amount of residual herbicides in Ag2197 was much lower than that in the non-transgenic plant, indicating that the transgenic plant metabolized the herbicides to a detoxified form. The transgenic plants produced in this study might be useful for the phytoremediation of chemical pollution in the soil.

Inducible cross-tolerance to herbicides in transgenic potato plants with the rat CYP1A1 gene.

A gene of the enzyme involved in xenobiotic metabolism in mammalian liver was introduced into potato to confer inducible herbicide tolerance. A rat cytochrome P450 monooxygenase, CYP1A1 cDNA, was kept under the control of the tobacco PR1a promoter in order to apply the system of chemical inducible expression using the plant activator Benzothiadiazole (BTH). Transgenic plants were obtained based on the kanamycin resistance test and PCR analysis. Northern-blot analysis revealed the accumulation of mRNA corresponding to rat CYP1A1 in the transgenic plants treated with BTH (3.0 micro mol/pot), whereas no accumulation of the corresponding mRNA occurred without BTH treatment. These transgenic plants also produced a protein corresponding to CYP1A1 in the leaves by BTH treatment. The transgenic plants with BTH application showed a much-higher tolerance to the phenylurea herbicides chlortoluron and methabenzthiazuron than non-transgenic plants. These findings indicated that the ability of metabolizing the two herbicides to less-toxic derivatives was displayed in the transgenic plants after BTH treatment. Transgenic plants harboring the CYP1A1 cDNA fused with the yeast P450 reductase (YR) gene under the control of PR1a were also produced. Although the plants showed a lower expression level of the fused gene than transgenic plants with CYP1A1 cDNA alone, they were tolerant to herbicides. These facts suggested that the CYP1A1 enzyme fused with YR showed a higher specific activity than CYP1A1 alone. This study demonstrated that the mammalian cDNA for the de-toxification enzyme of herbicides under the control of the PR1a promoter conferred chemical-inducible herbicide tolerance on potato.

The clinical importance of the trimethadione tolerance test as a method for quantitative assessment of hepatic functional reserve in patients with biliary atresia.

BACKGROUND: The trimethadione (TMO) tolerance test was performed to evaluate its usefulness in the assessment of hepatic functional reserve in patients with biliary atresia. METHOD: Nineteen patients with biliary atresia after hepatic portoenterostomy (age range: 2 months to 25 years; sex: 6 males and 13 females) were studied. The study was performed in the morning after a 12-h fast. TMO was given orally, at a dose of 4 mg/kg, with 5 mL of 5% glucose 2 h before breakfast. Blood samples (0.5 mL) were collected to determine serum TMO and dimethadione (DMO), a metabolite of TMO, levels 4 h after the administration of TMO. TMO and DMO were measured by a gas-liquid chromatographic method. RESULTS: A higher total bilirubin level (over 1 mg/dL) in patients with jaundice was reflected in the smaller serum DMO/TMO ratio 4 h after the oral administration of TMO. In addition, these patients with total bilirubin levels of 1 mg/dL or less had a significantly lower DMO/TMO ratio than the control group (healthy subjects). The serum DMO/TMO ratio showed a close correlation with the Child-Pugh score, which is used for overall evaluation of severity of cirrhosis and Mayo risk scores for primary biliary cirrhosis in adults (0.856, P < 0.01 and 0.788, P < 0.01, respectively). The TMO tolerance test shows the benefit of performing a relatively early test of dynamic liver function to evaluate hepatic functional reserve in pre- and post-operative biliary atresia patients.

Binding of annexins to lung lamellar bodies and the PMA-stimulated secretion of annexin V from alveolar type II cells.

To identify lung lamellar body (LB)-binding proteins, the fractions binding to LB-Sepharose 4B in a Ca(2+)-dependent manner from the lung soluble fractions were analyzed with Mono Q column. Four annexins (annexins III, IV, V, and VIII) were identified by partial amino acid sequence analyses as the LB-binding proteins in the lung soluble fractions. A control experiment using phospholipid (phosphatidylserine/phosphatidylglycerol/phosphtidylcholine) liposome-Sepharose 4B revealed that annexins III, IV and V were the Ca(2+)-dependent proteins binding to the column in the lung soluble fractions, while annexin VIII was not detected. Thus, annexin VIII might preferentially bind to LB. On the other hand, the only Ca(2+)-dependent LB-binding protein identified in the bronchoalveolar lavage fluids was annexin V. It was further demonstrated that annexin V was secreted by isolated alveolar type II cells from rats and that the secretion was stimulated by the addition of phorbol ester (PMA), a potent stimulator of surfactant secretion. The PMA-dependent stimulation of annexin V was attenuated by preincubation with surfactant protein-A (SP-A), a potent inhibitor of surfactant secretion. As LB is thought to be an intracellular store of pulmonary surfactant, which is secreted by alveolar type II cells, annexin V is likely to be secreted together with the lamellar body.

Distinct constitutive and low-CO2-induced CO2 uptake systems in cyanobacteria: genes involved and their phylogenetic relationship with homologous genes in other organisms.

Cyanobacteria possess a CO(2)-concentrating mechanism that involves active CO(2) uptake and HCO(3)(-) transport. For CO(2) uptake, we have identified two systems in the cyanobacterium Synechocystis sp. strain PCC 6803, one induced at low CO(2) and one constitutive. The low CO(2)-induced system showed higher maximal activity and higher affinity for CO(2) than the constitutive system. On the basis of speculation that separate NAD(P)H dehydrogenase complexes were essential for each of these systems, we reasoned that inactivation of one system would allow selection of mutants defective in the other. Thus, mutants unable to grow at pH 7.0 in air were recovered after transformation of a DeltandhD3 mutant with a transposon-bearing library. Four of them had tags within slr1302 (designated cupB), a homologue of sll1734 (cupA), which is cotranscribed with ndhF3 and ndhD3. The DeltacupB, DeltandhD4, and DeltandhF4 mutants showed CO(2)-uptake characteristics of the low CO(2)induced system observed in wild type. In contrast, mutants DeltacupA, DeltandhD3, and DeltandhF3 showed characteristics of the constitutive CO(2)-uptake system. Double mutants impaired in one component of each of the systems were unable to take up CO(2) and required high CO(2) for growth. Phylogenetic analysis indicated that the ndhD3/ndhD4-, ndhF3/ndhF4-, and cupA/cupB-type genes are present only in cyanobacteria. Most of the cyanobacterial strains studied possess the ndhD3/ndhD4-, ndhF3/ndhF4-, and cupA/cupB-type genes in pairs. Thus, the two types of NAD(P)H dehydrogenase complexes essential for low CO(2)-induced and constitutive CO(2)-uptake systems associated with the NdhD3/NdhF3/CupA-homologues and NdhD4/NdhF4/CupB-homologues, respectively, appear to be present in these cyanobacterial strains but not in other organisms.

Mapping loci causing susceptibility to anal atresia in pigs, using a resource pedigree.

BACKGROUND/PURPOSE: Very little information on the genetic background for anal atresia (anorectal malformations; AA) in humans has been described. A strikingly similar natural anomaly occurs in piglets. The authors have used this as an animal model for various research purposes. The affected piglets were treated surgically soon after birth, raised, and used for breeding. The authors have generated a resource pedigree segregating for this naturally occurring nonsyndromal AA and describe here the first attempt to map susceptibility loci by marker analysis. METHODS: A pig pedigree with a high incidence of AA has been established by selective breeding using 3 probands from the Landrace and Large White breeds. It has been maintained by intrafamilial crossing for more than 15 years. A backcross pedigree has now been generated by mating 4 AA females to an unaffected male from the Chinese Meishan breed. F(1) animals were both intercrossed and backcrossed to affected AA animals. A genome scan was carried out using the F(0), F(1), and affected backcross progeny. Ninety-two microsatellite loci were analyzed using fluorescently labelled primers and an ABI377 sequencer. Linkage analysis was done with the CRI-MAP 2.4 software. RESULTS: Crossing affected parents increased the incidence of abnormalities from 30% to 61.9%. All 39 F(1) pigs were unaffected. In the F(1) intercross, only 3 of 205 (1.5%) were affected, whereas 42 of 523 (8.0%) backcross progeny were affected. The marked difference in the incidence of affected progeny in the F(1) intercross and in the backcross indicates the presence of multiple genes causing AA. The genome scan showed suggestive evidence for the presence of a susceptibility locus on pig chromosome 15 (lod score 2.7 for a pig microsatellite marker SW2072). CONCLUSIONS: The results clearly show that AA has a oligogenic or polygenic background. The genome scan showed one suggestive locus causing AA on pig chromosome 15. The long-term goal is to identify causative genes for this malformation by comparative positional candidate cloning. This study provides, for the first time, linkage mapping of nonsyndromal anorectal malformations with a polygenic inheritance.

A monoclonal antibody-based ELISA for the analysis of the insecticide flucythrinate in environmental and crop samples.

A competitive enzyme-linked immunosorbent assay (ELISA) has been developed for the detection of the insecticide flucythrinate in environmental and food samples. Two types of haptens, the acid moiety that is the hydrolyzed product of flucythrinate, and the carboxylated propyl derivative of the alcohol moiety, were used to prepare monoclonal antibodies (MAbs). Five MAbs, which raised against the former hapten, were reactive with flucythrinate. Among them, MAb F1A27-4 showed the highest activity toward flucythrinate, and did not cross-react with other pyrethroids such as cycloprothrin, fenvalerate, fluvalinate, etofenprox and silafluofen. The assay conditions of indirect competitive ELISA with MAb F1A27-4 were studied to optimize the detection of flucythrinate in environmental and food samples. Incubation at 4 degrees C in the assay buffer, pH 8, with 300 mM sodium chloride improved the sensitivity. The addition of rabbit serum albumin or rabbit antiserum and the presence of 50 ml litre-1 of methanol reduced matrix effects of the samples. Under optimized conditions, the ELISA detected flucythrinate spiked in water, soil, and extracts of apple and tea samples down to 10 mg litre-1, 0.2 mg litre-1, 0.3 mg litre-1 and 0.3 mg litre-1, respectively. The mean recovery and CV ranged from 91% to 120% and from 5% to 12%, respectively. The ELISA results in apple samples correlated well with those from LC-MS analysis (r2 = 0.99, n = 12).

Localization of NAD(P)H dehydrogenase in the cyanobacterium Synechocystis sp. strain PCC 6803.

Antibodies raised against NdhH and NdhB detected these proteins in the thylakoid membrane of Synechocystis sp. strain PCC 6803, but not in a purified cytoplasmic membrane. We conclude that NAD(P)H dehydrogenase is largely, if not exclusively, confined to the thylakoid membrane.

Increased noradrenaline release from rat preoptic area during and after sevoflurane and isoflurane anesthesia.

PURPOSE: To study the effects of sevoflurane and isoflurane on noradrenaline release from the rat preoptic area (POA). METHOD: Sixteen male Wistar rats were studied. A microdialysis probe with a 2 mm long semipermeable membrane was implanted in the POA. Dialysates were collected at intervals often minutes. After obtaining five control samples for 50 min, 30 min inhalation of 3% sevoflurane or 1.8% isoflurane was performed. After cessation of the inhalation, five more samples were obtained for 50 min as recovery phase. Noradrenaline (NA) concentration in the dialysates was measured by high pressure liquid chromatography with an electrochemical detector. RESULTS: Both sevoflurane and isoflurane caused marked increases in NA release from the rat POA (sevoflurane 233% at 20 min, isoflurane 357% at ten minutes after the start of inhalation). The marked NA releases were also observed during the emergence from sevoflurane and isoflurane anesthesia (sevoflurane 269% at 20 min, isoflurane 368% at ten minutes in the recovery phase). CONCLUSION: This study suggests that enhanced release of NA in the POA during sevoflurane and isoflurane may explain the excitatory phase observed during the peri-anesthetic period with these agents.

Alteration of annexin IV expression in alcoholics.

Western blot analysis was performed by using a specific antibody to measure annexin IV in human postmortem brain samples from alcoholic subjects. The analysis showed a significantly augmented expression in the hippocampus compared with controls, whereas the expression in the frontal cortex was equivalent in both groups. Annexin IV expression in the occipital cortex tended to increase in alcoholics. It was shown further that autoantibodies to annexin IV were increased significantly in alcoholic patients compared with controls. Thus, annexin IV may become a novel biological marker for alcoholics.

Differential lipid specificities of the repeated domains of annexin IV.

The roles of the four domains of annexin IV in binding to phospholipids and glycolipids were assessed by analyzing the binding of a group of mutant annexins IV in which one or more of the four domains was inactivated by replacing a critical amino residue(s) (Asp or Glu) with the neutral residue Ala. The data reveal that individual annexin domains may have characteristic affinities for different lipids. In particular, inactivation of the fourth domain inhibits the binding to phosphatidylserine (PS) and phosphatidylinositol (PI) but not to phosphatidylglycerol (PG), suggesting that this domain specifically can accommodate the larger head groups of PS and PI whereas the other three domains may form more restricted binding pockets. In order to block binding to PG, domain 1, or both domains 2 and 3 must be inactivated in addition to domain 4, suggesting that all four domains may be able to accommodate the headgroup of PG to some extent. Binding to acidic glycolipids (sulfatides) was also sensitive to inactivation of domain 4. However, in the case of sulfatides the nature of the binding reaction is fundamentally different compared with the binding to phospholipids since the interaction with sulfatides was highly sensitive to an increase in ionic strength. The binding to sulfatides may depend therefore on charge-charge interactions whereas the binding to phospholipid may involve a more specific interaction between the lipid headgroup and the protein surface, and/or interaction of the protein with the hydrophobic portion of a lipid bilayer.

Expression of human cytochromes P450 1A1 and P450 1A2 as fused enzymes with yeast NADPH-cytochrome P450 oxidoreductase in transgenic tobacco plants.

Among 11 isoforms of the human cytochrome P450 enzymes metabolizing xenobiotics, CYP 1A1 and CYP 1A2 were major P450 species in the metabolism of the herbicides chlortoluron and atrazine in a yeast expression system. CYP1A2 was more active in the metabolism of both herbicides than CYP1A1. The fused enzymes of CYP1A1 and CYP1A2 with yeast NADPH-cytochrome P450 oxidoreductase were functionally active in the microsomal fraction of the yeast Saccharomyces cerevisiae and showed increased specific activity towards 7-ethoxyresorufin as compared to CYP1A1 and CYP1A2 alone. Then, both fused enzymes were each expressed in the microsomes of tobacco (Nicotiana tabacum cv. Samsun NN) plants. The transgenic plants expressing the CYP1A2 fusion enzyme had higher resistance to the herbicide chlortoluron than the plants expressing the CYP1A1 fusion enzyme did. The transgenic plants expressing the CYP1A2 fused enzyme metabolized chlortoluron to a larger extent to its non-phytotoxic metabolites through N-demethylation and ring-methyl hydroxylation as compared to the plants expressing the CYP1A1 fused enzyme. Thus, the possibility of increasing the herbicide resistance in the transgenic plants by the selection of P450 species and the fusion with P450 reductase is discussed.

Synthesis of multiacyl poly(ethylene glycol) for the conjugation of cytochrome c to phospholipid vesicle.

To conjugate water-soluble macromolecules on the surface of phospholipid vesicles, we synthesized a poly(ethylene glycol) (PEG)-lipid having four acyl chains using a lysine (Lys)-type monodendron structure. One end of the diamino-PEG was amidified with Lys, and then two amino groups of the Lys moiety were amidified with two Lys derivatives which had been acylated with two stearoyl groups. The other end of the PEG was activated with a triazine group or a pyridyldithio group. The hydrate of the lipid mixture of dipalmitoylphosphatidylcholine, cholesterol, dipalmitoylphosphatidylglycerol, and the PEG-lipid at a molar ratio of 5/5/1/0.3 was extruded in order to prepare the phospholipid vesicles with the average diameter of 270 +/- 20 nm. The coupling ratio of cytochrome c with the PEG-lipid was monitored by HPLC, detecting the pyridyl 2-thione liberated from the pyridyldithio group and determining it to be 26% on the basis of the incorporated PEG-lipid.

Biphasic kinetic behavior of rat cytochrome P-4501A1-dependent monooxygenation in recombinant yeast microsomes.

Rat cytochrome P-4501A1-dependent monooxygenase activities were examined in detail using recombinant yeast microsomes containing rat cytochrome P-4501A1 and yeast NADPH-P-450 reductase. On 7-ethoxycoumarin, which is one of the most popular substrates of P-4501A1, the relationship between the initial velocity (v) and the substrate concentration ([S]) exhibited non-linear Michaelis-Menten kinetics. Hanes-Woolf plots ([S]/v vs. [S]) clearly showed a biphasic kinetic behavior. Aminopyrine N-demethylation also showed a biphasic kinetics. The regression analyses on the basis of the two-substrate binding model proposed by Korzekwa et al. (Biochemistry 37 (1998) 4137-4147) strongly suggest the presence of the two substrate-binding sites in P-4501A1 molecules for those substrates. An Arrhenius plot with high 7-ethoxycoumarin concentration showed a breakpoint at around 28 degrees C probably due to the change of the rate-limiting step of P-4501A1-dependent 7-ethoxycoumarin O-deethylation. However, the addition of 30% glycerol to the reaction mixture prevented observation of the breakpoint. The methanol used as a solvent of 7-ethoxycoumarin was found to be a non-competitive inhibitor. Based on the inhibition kinetics, the real V(max) value in the absence of methanol was calculated. These results strongly suggest that the recombinant yeast microsomal membrane containing a single P-450 isoform and yeast NADPH-P-450 reductase is quite useful for kinetic studies on P-450-dependent monooxygenation including an exact evaluation of inhibitory effects of organic solvents.

CYP78A1 preferentially expressed in developing inflorescences of Zea mays encoded a cytochrome P450-dependent lauric acid 12-monooxygenase.

Cytochrome P450 (P450 or CYP) monooxygenases play an important role in the oxidation of a number of lipophilic substrates including secondary metabolites in higher plants. Larkin reported that CYP78A1 was preferentially expressed in developing inflorescences of Zea mays (Larkin, Plant Mol. Biol. 25: 343-353, 1994). However, the enzymatic function of CYP78A1 hasn't been clarified yet. To characterized the enzymatic activity of CYP78A1, in this study, CYP78A1 cDNA and tobacco or yeast NADPH-cytochrome P450 oxidoreductase (P450 reductase) was expressed in the yeast Saccharomyces cerevisiae AH22 cells under the control of alcohol dehydrogenase promoter I and terminator. The reduced CO-difference spectrum of a microsomal fraction prepared from the transformed yeast cells expressing CYP78A1 and yeast P450 reductase showed a peak at 449 nm. Based on the spectrum, the content of a P450 molecule was estimated to be 45 pmol P450 equivalent/ mg of protein in the microsomal fraction. The recombinant yeast microsomes containing CYP78A1 and yeast P450 reductase were found to catalyze 12-monooxygenation of lauric acid. Based on these results, CYP78A1 preferentially expressed in developing inflorescences of Zea mays appeared to have participated in the monooxygenation of fatty acids.