Prediction of clinical bacteriological efficacy of oral antibiotics using a mechanism-based pharmacokinetic-pharmacodynamics modeling.

The objective of this study was to predict the clinical bacteriological efficacy of antibiotics and to examine the pharmacodynamics (PD) characteristics of antibiotics against bacterial strains using a mechanism-based pharmacokinetic-pharmacodynamics (PK-PD) modeling developed on the basis of interaction between drug concentrations and antibacterial activities. Dynamic PD parameters (epsilon, gamma, EC50) and growth rate of organisms (lambda) were obtained from in vitro time-kill profile data of oral antibiotics, tebipenem pivoxil (TBPM-PI) and cefditoren pivoxil (CDTR-PI) against Streptococcus pneumoniae or Haemophilus influenzae. PD characteristics of both drugs against S. pneumoniae or H. influenzae were examined, which indicated TBPM was concentration-dependent as well as time-dependent, and CDTR was mainly time-dependent to exhibit their bactericidal activities. Next, we simulated TBPM and CDTR concentrations in plasma after oral administration according to the dosage regimen of each drug specified in package insert, using population pharmacokinetic parameters of both drugs in pediatric patients with infections. In addition, changes in viable in vivo bacterial counts in humans were simulated using dynamic PD parameters and mean plasma concentrations of each drug. As a result, simulated profile of viable counts of S. pneumoniae and H. influenzae were well corresponding to the bacteriological efficacy results in clinical double-blinded comparative study of TBPM-PI and CDTR-PI in oral administration to pediatric patients with acute otitis media. As mentioned in the above, it was considered to be possible to clarify the PD characteristics of TBPM and CDTR against each bacterial strain using the mechanism-based PK-PD model developed on the basis of interaction between drug concentrations and antibacterial activities, and to estimate the clinical bacteriological efficacy of those drugs.

Population pharmacokinetics of cefditoren pivoxil in non-infected adults.

Population pharmacokinetic analysis was conducted on cefditoren pivoxil (CDTR-PI), a third generation oral antibiotic to evaluate the effect of covariates on pharmacokinetic parameters. Plasma concentrations of cefditoren (CDTR, total number of sampling points: 2864) obtained from healthy adult subjects, elderlies, and subjects with renal dysfunction (287 subjects) after CDTR-PI administration as well as demographic data of those subjects were used for analysis. We conducted the population pharmacokinetic analysis of CDTR-PI using a nonlinear mixed effects modeling (NONMEM) method. A one-compartment model with a first-order absorption and lag time fitted well to plasma concentration-time curve for CDTR. The subject covariate significantly affecting pharmacokinetic parameters of CDTR-PI was demonstrated by population pharmacokinetic analysis. The absorption rate constant (ka: hr(-1)) of CDTR-PI decreased with age, total clearance adjusted by bioavailability (CL/F: L/hr/kg) increased with increasing creatinine clearance adjusted by body weight (Ccr: mL/min/kg) and volume of distribution adjusted by bioavailability (Vd/F: L/kg) decreased with increasing body weight (WT: kg). In addition, the lag time (Tlag: hr) depends on formulation (tablet or granule) of CDTR-PI and the absorption lag time of the tablet was longer than that of the granule. We could obtain the population mean parameters of CDTR-PI together with interindividual variability and intraindividual residual variability after oral administration of CDTR-PI to adult subjects. In the future, this information will enable us to simulate the plasma concentrations of CDTR in subjects with various demographic backgrounds, which contributes to future examination of the efficacy and safety of CDTR-PI.

Population pharmacokinetic analysis of cefditoren pivoxil in pediatric patients with infection.

Population pharmacokinetic analysis was conducted on cefditoren pivoxil (CDTR-PI, Brand name: MEIACT, Meiji Seika Pharma Co., Ltd.), a third generation oral antibiotic, using plasma concentrations of cefditoren (CDTR, total number of sampling points: 578) obtained from pediatric patients (153 subjects, dose: 5.62 +/- 1.62 mg/kg) after CDTR-PI administration as well as demographic data of those subjects. NONMEM (Ver. VI LEVEL 2.0) was used as software. The first-order conditional estimation (FOCE) method without interaction was employed as algorithm. A one-compartment model with first-order absorption was used as a pharmacokinetic model. As the result of analysis, the following population pharmacokinetic parameters were obtained for CDTR. Population mean parameters: ka (hr(-1)) = 0.527, CL/F (L/hr/kg) = -0.474 x Scr + 0.82, Vd/F (L/kg) = 0.77, Tlag (hr) = 0.282 x (1+0.435 x NAT) (NAT: 0 = Japan, 1 = USA, interindividual variability: omega (ka) = 17.23%, omega (CL/F) = 33.02%, omega (Vd/F) = 86.66%, intraindividual residual variability: sigma = 0.428 microg/mL. Bayes estimation was carried out for each subject using the final model to calculate secondary parameters such as C(max), T(max), AUC, and t1/2. C(max) and AUC increased significantly with dose. However, T(max) was approximately 2hours and t1/2 was approximately 1 hour at any dose level, showing no significant dose-dependent changes. When CDTR-PI was administered orally to a child, a significant increase was noted in plasma CDTR concentrations, suggesting high efficacy. In addition, pharmacokinetics of CDTR were simulated in patients with renal impairment using the final model. As a result, a delay in T(max) and increases in AUC, C(max), and t1/2 were presumed with increased Scr, and the degrees of such increases were also quantitatively estimated. As mentioned above, the population pharmacokinetic parameters of CDTR were obtained, which is sure contribute to simulation of its plasma concentrations in patients with various backgrounds and to speculation of its efficacy and safety.

Selection of Cord Blood Unit by CD34+ Cell and GM-CFU Numbers and Allele-Level HLA Matching in Single Cord Blood Transplantation.

In Japan, only single-unit cord blood transplantations (CBTs) are typically performed, and their number has increased over the last 23 years, with ongoing improvement in results. In most cases, CBTs with multiple HLA mismatches are used, owing to a low HLA barrier, and lower engraftment rate is a problem that must be overcome. Here, as part of an effort to improve guidelines for the selection and processing of CB units for transplantation, we sought to assess the present status of CBT in Japan and to elucidate factors contributing to the favorable outcomes, focusing in particular on selection by cell components of CB unit and HLA allele matching. We conducted a nationwide study analyzing 13,443 patients who underwent first CBT between in Japan between December 1997 and December 2019 using multivariate regression analysis. Both patient- and transplantation-related variables, such as age and Hematopoietic Cell Transplantation Comorbidity Index, as well as selected CB unit characteristics, were included in the analysis. The interaction analysis elucidated that CB unit selection favoring higher counts of CD34+ cells and granulocyte macrophage colony-forming units (GM-CFU)/kg, but not of total nucleated cells, contributed to improved engraftment after transplantation. Moreover, a higher CD34+ cell dose was associated with improved overall survival (OS). Distinctive HLA allele matching was observed. A 0 or 1 HLA allele mismatch between patient and donor had favorable engraftment and carried significantly lower risks of acute GVHD and chronic GVHD but had a significantly higher leukemia relapse rate, compared with a 3-HLA allele mismatch. HLA-DRB1 mismatches were associated with reduced risk of leukemia relapse. Notably, the number of HLA allele mismatches had no incremental effect on engraftment, acute and chronic GVHD, or relapse incidence. As a result, 5-year overall survival did not differ significantly among patients receiving CB units with 0 to 7 HLA allele mismatches. The main points of CB unit selection are as follows. First, selection according to a higher number of CD34+ cells/kg and then of CFU-GM/kg is recommended to obtain favorable engraftment. A unit with .5 × 105 CD34+ cells/kg is minimally acceptable. For units with a CD34+ cell dose of .5 to 1.0 × 105 cells/kg, applying the parameter of ≥20 to 50 × 103 GM-CFU/kg (66.5% of transplanted CB units in this cohort) is associated with a neutrophil engraftment rate of approximately 90%. A unit with ≥1.0 × 105 CD34+ cells/kg can achieve a ≥90% mean neutrophil engraftment rate. Subsequently, HLA allele matching of HLA-A, -B, -C, and -DRB1 at the 2-field level should be searched for units with 0 or 1 HLA allele mismatch in the host-versus-graft direction for favorable engraftment. Units with 2 to 6 HLA allele mismatches are acceptable in patients age ≥15 years and units with 2 to 4 HLA allele mismatches are acceptable in patients age ≤14 years. Units with HLA-DRB1 and/or -B allele mismatch(es) might not be preferable owing to an increased GVHD risk. Our analysis demonstrates that single-unit CBT with the selection of adequate CD34+/kg and GM-CFU/kg and HLA allele matching showed favorable outcomes in both pediatric and adult patients.

High throughput method of 16S rRNA gene sequencing library preparation for plant root microbial community profiling.

Microbiota are a major component of agroecosystems. Root microbiota, which inhabit the inside and surface of plant roots, play a significant role in plant growth and health. As next-generation sequencing technology allows the capture of microbial profiles without culturing the microbes, profiling of plant microbiota has become a staple tool in plant science and agriculture. Here, we have increased sample handling efficiency in a two-step PCR amplification protocol for 16S rRNA gene sequencing of plant root microbiota, improving DNA extraction using AMPure XP magnetic beads and PCR purification using exonuclease. These modifications reduce sample handling and capture microbial diversity comparable to that obtained by the manual method. We found a buffer with AMPure XP magnetic beads enabled efficient extraction of microbial DNA directly from plant roots. We also demonstrated that purification using exonuclease before the second PCR step enabled the capture of higher degrees of microbial diversity, thus allowing for the detection of minor bacteria compared with the purification using magnetic beads in this step. In addition, our method generated comparable microbiome profile data in plant roots and soils to that of using common commercially available DNA extraction kits, such as DNeasy PowerSoil Pro Kit and FastDNA SPIN Kit for Soil. Our method offers a simple and high-throughput option for maintaining the quality of plant root microbial community profiling.

Structural and spectroscopic characterization of CO inhibition of [NiFe]-hydrogenase from Citrobacter sp. S-77.

Hydrogenases catalyze the reversible oxidation of H2. Carbon monoxide (CO) is known to be a competitive inhibitor of O2-sensitive [NiFe]-hydrogenases. Although the activities of some O2-tolerant [NiFe]-hydrogenases are unaffected by CO, the partially O2-tolerant [NiFe]-hydrogenase from Citrobacter sp. S-77 (S77-HYB) is inhibited by CO. In this work, the CO-bound state of S77-HYB was characterized by activity assays, spectroscopic techniques and X-ray crystallography. Electron paramagnetic resonance spectroscopy showed a diamagnetic Ni2+ state, and Fourier-transform infrared spectroscopy revealed the stretching vibration of the exogenous CO ligand. The crystal structure determined at 1.77 Šresolution revealed that CO binds weakly to the nickel ion in the Ni-Fe active site of S77-HYB. These results suggest a positive correlation between O2 and CO tolerance in [NiFe]-hydrogenases.

Combined impact of HLA-allele matching and the CD34-positive cell dose on optimal unit selection for single-unit cord blood transplantation in adults.

The combined effects of HLA-allele matching at six-loci (HLA-A, -B, -C, -DRB1, -DQB1, and -DPB1) and CD34+ cell dose on clinical outcomes were analyzed in 1,226 adult cases with single-unit unrelated cord blood transplantation. In the six-loci analysis, low HLA-allele matches did not significantly increase the overall mortality compared to higher matches, whereas in the five-loci analysis excluding HLA-DPB1, they caused a higher overall mortality (HR 1.42, p = .002), possibly due to the graft-versus-leukemia effect of HLA-DPB1 mismatches. A lower CD34+ cell dose (<.50 × 105/kg) resulted in higher mortality and lower engraftment; these inferior outcomes were offset by high HLA-allele matches (7-10/10 match), while the inferior outcomes of low HLA-allele matches were improved by increasing the CD34+ cell dose. Consideration of the combined effects of the CD34+ cell dose and HLA matching may expand the options for transplantable units when HLA matching or the CD34+ cell dose is inadequate.

Risk of HLA Homozygous Cord Blood Transplantation: Implications for Induced Pluripotent Stem Cell Banking and Transplantation.

Clinical application of induced pluripotent stem cells (iPS) in autologous settings has just begun. To overcome the high time and cost barriers in the individual production of autologous iPS, the use of allogeneic iPS with a homozygous human leukocyte antigen (HLA) haplotype (HLA-homo HP) has been proposed. Cord blood transplantation (CBT) is a suitable model for evaluating the allogeneic immunogenicity of iPS transplantation from HLA-homo donors. We analyzed 1,374 Japanese single cord blood transplant pairs who were retrospectively typed as HLA-A, -B, -C, -DRB1, -DQB1, and -DPB1. Among these, six pairs with donor HLA homo-patient-HLA hetero (homo-hetero) were found, all of which showed favorable neutrophil engraftment. Multivariate analysis revealed a significantly elevated engraftment risk (HR = 1.59) compared with hetero-hetero pairs with HLA 1-2 locus mismatch (789 pts) and comparative risk (HR = 1.23) compared with hetero-hetero pairs with 0 mismatch (104 pts). These results for CBT with HLA-homo HP cord blood carry an important implication, namely the possibility that HLA-homo iPS transplantation results in favorable engraftment. Furthermore, we obtained detailed information on HLA alleles and haplotypes of HLA-homo. All donor HLA-homo HPs had a common specific ethnicity and high conservation of the HLA region, and one of two patient heterogeneous HPs invariably shared the same HP as donor HLA-homo HP, and another non-shared patient HP was mismatched with 1 to 4 HLA alleles of HLA-A, -B, -C, and -DRB1 loci in the GVH direction. These findings indicate that patients possessing a single common HLA haplotype have a higher chance of yielding HLA-homo iPS. Stem Cells Translational Medicine 2018;7:173-179.

Downregulation of drs mRNA expression is associated with the progression of adult T-cell leukemia/lymphoma.

Although adult T-cell leukemia/lymphoma (ATLL) is initiated by infection with human T-cell leukemia virus (HTLV-1), many other factors are thought to be required for the progression from indolent ATL to aggressive ATLL. The drs gene was originally isolated as a novel suppressor gene of v-src transformation and was shown to induce apoptosis in human cancer cells. To investigate the involvement of drs downregulation in the progression of ATLL, we examined the expression of drs in smoldering, chronic and aggressive ATLL, and found that drs expression was markedly reduced in clinically aggressive ATLL. In aggressive ATLL cell lines, expression of drs mRNA was not detected, although expression of drs mRNA was detected in T-cell lines infected with HTLV-1. A correlation between drs downregulation and expression of the Tax gene was not observed in these T-cell lines. Furthermore, introduction of drs into an ATL cell line, HUT102, by retrovirus vector suppressed the colony formation of the cells in soft agar and enhanced apoptotic cell death of the cells under low serum culture conditions. These results indicate that downregulation of drs is closely linked to the progression of ATLL, independently of Tax expression, suggesting that drs may suppress the progression of ATLL via enhancing apoptosis.

HIV-1-derived self-inactivating lentivirus vector induces megakaryocyte lineage-specific gene expression.

Pluripotent, self-renewing, hematopoietic stem cells are considered good targets for gene modification to treat a wide variety of disorders. However, as many genes are expressed in a stage-specific manner during the course of hematopoietic development, it is necessary to establish a lineage-specific gene expression system to ensure the proper expression of transduced genes in hematopoietic stem cells. In this study, we constructed a VSV-G-pseudotyped, human immunodeficiency virus type 1-based, self-inactivating lentivirus vector that expressed green fluorescent protein (GFP) under the control of the human CD41 (glycoprotein 2b; GP2b) promoter; this activity is restricted to megakaryocytic lineage cells. The recombinant virus was used to infect human peripheral blood CD34+ (hematopoietic stem/progenitor) cells, and lineage-specific gene expression was monitored with GFP measurements. The analysis by FACS determined that GFP expression driven by the GP2b promoter was restricted to megakaryocytic progenitors and was not present in erythrocytes. Furthermore, in the hematopoietic colony-forming assay, GFP expression was restricted to colony-forming units-megakaryocyte (CFU-Meg) colonies under the control of the GP2b promoter, whereas all myeloid colonies (burst-forming units-erythroid, colony-forming units-granulocyte-macrophage, and CFU-Meg) expressed GFP when the transgene was regulated by the cytomegalovirus promoter. These results demonstrated lineage-specific expression after gene transduction of hematopoietic stem cells. The application of this vector system should provide a useful tool for gene therapy to treat disorders associated with megakaryocyte (platelet) dysfunction.

Expression and affinity of homing-related molecules on steady-state adult and neonate human PB CD34+ cells and their SRC activity.

Although the vast majority of hematopoietic progenitor cells (HPCs) reside within the bone marrow (BM), a small number of HPCs also continuously circulate in the peripheral blood (PB). The examination of the fate of blood-borne HPCs in parabiotic mice, which are surgically conjoined and share a common circulation, recently revealed that steady-state PB HPCs play a physiological role in, at least, the functional re-engraftment of unconditioned BM. To assess the possibility that human HPCs have a similar function, in this study we examined the expression level and affinity of the homing-related molecules, as well as the SCID mouse reconstituting cell (SRC) activity of human PB CD34+ cells, and compared adults with neonates. There was no remarkable difference between adults and neonates in the expression of E- and/or P-selectin ligands by PB CD34+ cells or in these cells' affinity to VCAM-1. In contrast, the expression level of CXCR4 on PB CD34+ cells was much lower in adults than in neonates. Adult cells also showed a much lower SRC activity than neonates. These results suggest that human PB HPCs may contribute to steady-state hematopoiesis in the BM of neonates to some extent, but not so much in adults.

Spontaneous and rapid reexpression of functional CXCR4 by human steady-state peripheral blood CD34+ cells.

Although only 5% of steady-state peripheral blood (PB) CD34+ cells were found to express chemokine receptor CXCR4, 45% of the cells became CXCR4+ after incubation at 37 degrees C for 4 hours. In contrast, there were no remarkable differences between PB CD34+ cells before and after the 37 degrees C incubation in their expression of selectin ligand, VLA-4, and VLA-5 or in their affinity for VCAM-1 or fibronectin. This increase in CXCR4 expression level was inhibited by the addition of brefeldin A, actinomycin D, or cycloheximide. When PB CD34+ cells with CXCR4 expression levels enhanced by a 4-hour preincubation at 37 degrees C or bone marrow (BM) CD34+ cells were exposed overnight to stromal cell-derived factor 1 (SDF-1), the expression levels of CXCR4 were greatly reduced, and when SDF-1 was removed, CXCR4 levels were thereafter up-regulated. The reexpressed CXCR4 was able to elicit integrin-dependent migration of hematopoietic progenitor cells. There was no difference in the severe combined immunodeficient mouse repopulating cell activity between PB CD34+ cells with and cells without a 37 degrees C preincubation.

[The revised Japanese pharmaceutical law and research ethics: risk-benefit assessment of fetal stem cell research].

The Japanese Pharmaceutical Law was revised at the end of July 2002. The important features of this revision are the postmarketing safety scheme, especially for biological products, and reconstruction of the legislation for effective pharmaceutical development. This is based on the national policy to foster life sciences such as genetic research and regenerative medicine for both healthcare improvement and industrial promotion. Such research requires study participants who donate human tissue including abandoned embryos or aborted fetuses, which may touch the human dignity. In particular, fetal stem cell research appears to have unpredictable risks not only to women who undergo abortions but also to societal epistemology. The authors conducted risk-benefit assessment of fetal stem cell research, reviewing the scientific, ethical, legal, and social aspects, including a case study of critical appraisal on a report of the double-blind, sham surgery-controlled trial of implantation of fetal tissues in patients with Parkinson's disease conducted in the USA. It is concluded that risk-benefit assessment with a wide, profound perspective is necessary for advanced biotechnology. Some types of research should not be assessed based only on such utilitarian viewpoints as risk and benefit. Conscientious reflection is necessary to reach a public consensus on which types of human material can be utilized as research or pharmaceutical resources.

A survey on low-dose oral contraceptive transactions at pharmacies.

Low-dose oral contraceptives (OCs) were approved by the Japanese Ministry of Health, Labor and Welfare in 1999. However, few women use low-dose OCs (married 1.9%, unmarried 0.7%). A survey of pharmacies was conducted to determine the current status of OC transactions. Of the 449 pharmacies randomly selected from each prefecture in Japan, 408 agreed to be interviewed. The survey results indicated that few pharmacies (15.1% of the total) stocked low-dose OCs. Even among pharmacies where they were available, only a few (13.5%) offered a wide variety of low-dose OCs for immediate dispensing. The price of low-dose OCs varied widely (yen 1,167-yen 7,000). In many pharmacies, the amount of space and interior structure were not adequate for users to seek advice on and receive low-dose OCs. The survey revealed that the current environment of many pharmacies is not adequate for users to visit, consult on OC use, and receive their prescriptions. To promote wider use of low-dose OCs, the facilities of pharmacies as well as the behavior of pharmacists need to be improved to safeguard the privacy of users.

Multiple ETS family proteins regulate PF4 gene expression by binding to the same ETS binding site.

In previous studies on the mechanism underlying megakaryocyte-specific gene expression, several ETS motifs were found in each megakaryocyte-specific gene promoter. Although these studies suggested that several ETS family proteins regulate megakaryocyte-specific gene expression, only a few ETS family proteins have been identified. Platelet factor 4 (PF4) is a megakaryocyte-specific gene and its promoter includes multiple ETS motifs. We had previously shown that ETS-1 binds to an ETS motif in the PF4 promoter. However, the functions of the other ETS motifs are still unclear. The goal of this study was to investigate a novel functional ETS motif in the PF4 promoter and identify proteins binding to the motif. In electrophoretic mobility shift assays and a chromatin immunoprecipitation assay, FLI-1, ELF-1, and GABP bound to the -51 ETS site. Expression of FLI-1, ELF-1, and GABP activated the PF4 promoter in HepG2 cells. Mutation of a -51 ETS site attenuated FLI-1-, ELF-1-, and GABP-mediated transactivation of the promoter. siRNA analysis demonstrated that FLI-1, ELF-1, and GABP regulate PF4 gene expression in HEL cells. Among these three proteins, only FLI-1 synergistically activated the promoter with GATA-1. In addition, only FLI-1 expression was increased during megakaryocytic differentiation. Finally, the importance of the -51 ETS site for the activation of the PF4 promoter during physiological megakaryocytic differentiation was confirmed by a novel reporter gene assay using in vitro ES cell differentiation system. Together, these data suggest that FLI-1, ELF-1, and GABP regulate PF4 gene expression through the -51 ETS site in megakaryocytes and implicate the differentiation stage-specific regulation of PF4 gene expression by multiple ETS factors.

Down-regulation of ASY/Nogo transcription associated with progression of adult T-cell leukemia/lymphoma.

Adult T-cell leukemia/lymphoma (ATLL) is an aggressive form of human leukemia/lymphoma. Although this disease is initiated by infection with human T-lymphotropic virus type 1 (HTLV-1), many HTLV-1 carriers survive for a long period without aggressive illness, suggesting that other factors may play roles in the progression of ATLL to an aggressive state. However, the mechanism involved in this progression still remains unclear. Previously, we have reported that ASY/Nogo mRNA was markedly down-regulated in human small-cell lung carcinomas, whereas it was expressed in normal tissues and other lung carcinomas, such as adenocarcinoma and squamous cell carcinoma. To understand whether or not ASY/Nogo gene is involved in the progression of ATLL, we examined the expression of ASY/Nogo mRNA in smoldering, chronic and aggressive ATLL, and found that the expression level of ASY/Nogo mRNA was markedly reduced in clinically aggressive ATLL. HTLV-1 Tax expression was not affected by the down-regulation of ASY/Nogo mRNA. These results indicate that the ASY/Nogo gene may act as a suppressor against ATLL progression, independent of Tax expression.

Differences between peripheral blood and cord blood in the kinetics of lineage-restricted hematopoietic cells: implications for delayed platelet recovery following cord blood transplantation.

Cord blood (CB) cells are a useful source of hematopoietic cells for transplantation. The hematopoietic activities of CB cells are different from those of bone marrow and peripheral blood (PB) cells. Platelet recovery is significantly slower after transplantation with CB cells than with cells from other sources. However, the cellular mechanisms underlying these differences have not been elucidated. We compared the surface marker expression profiles of PB and CB hematopoietic cells. We focused on two surface markers of hematopoietic cell immaturity, i.e., CD34 and AC133. In addition to differences in surface marker expression, the PB and CB cells showed nonidentical differentiation pathways from AC133(+)CD34(+) (immature) hematopoietic cells to terminally differentiated cells. The majority of the AC133(+)CD34(+) PB cells initially lost AC133 expression and eventually became AC133(-)CD34(-) cells. In contrast, the AC133(+)CD34(+) CB cells did not go through the intermediate AC133(-)CD34(+) stage and lost both markers simultaneously. Meanwhile, the vast majority of megakaryocyte progenitors were of the AC133(-)CD34(+) phenotype. We conclude that the delayed recovery of platelets after CB transplantation is due to both subpopulation distribution and the process of differentiation from AC133(+)CD34(+) cells.

First total synthesis of (+/-)-Linderol A, a tricyclic hexahydrodibenzofuran constituent of Lindera umbellata bark, with potent inhibitory activity on melanin biosynthesis of cultured B-16 melanoma cells.

The first total synthesis of (+/-)-Linderol A, a hexahydrodibenzofuran constituent of Lindera umbellata bark, with potent inhibitory activity on the melanin biosynthesis of cultured B-16 melanoma cells, was achieved through 19 steps of reaction in 6.6% overall yield, in which the critical step was a tandem reaction of a 3-ethoxycarbonylcoumarin derivative with dimethylsulfoxonium methylide to yield the 2-ethoxycarbonylcyclopenta[b]benzofuran-3-ol derivative.

Alternative promoter identified between a hypermethylated upstream region of repetitive elements and a CpG island in human ABO histo-blood group genes.

We have studied the expression of human histo-blood group ABO genes during erythroid differentiation, using an ex vivo culture of AC133(-)CD34(+) cells obtained from peripheral blood. 5'-Rapid amplification of cDNA ends analysis of RNA from those cells revealed a novel transcription start site, which appeared to mark an alternative starting exon (1a) comprising 27 bp at the 5'-end of a CpG island in ABO genes. Results from reverse transcription-PCR specific to exon 1a indicated that the cells of both erythroid and epithelial lineages utilize this exon as the transcription starting exon. Transient transfection experiments showed that the region just upstream from the transcription start site possesses promoter activity in a cell type-specific manner when placed 5' adjacent to the reporter luciferase gene. Results from bisulfite genomic sequencing and reverse transcription-PCR analysis indicated that hypermethylation of the distal promoter region correlated with the absence of transcripts containing exon 1a, whereas hypermethylation in the interspersed repeats 5' adjacent to the distal promoter was commonly observed in all of the cell lines examined. These results suggest that a functional alternative promoter is located between the hypermethylated region of repetitive elements and the CpG island in the ABO genes.