Betulinic acid inhibits high-fat diet-induced obesity and improves energy balance by activating AMPK.

BACKGROUND AND AIM: Metabolic syndromes are prevalent worldwide and result in various complications including obesity, cardiovascular disease and type II diabetes. Betulinic acid (BA) is a naturally occurring triterpenoid that has anti-inflammatory properties. We hypothesized that treatment with BA may result in decreased body weight gain, adiposity and hepatic steatosis in a diet-induced mouse model of obesity. METHODS AND RESULTS: Mice fed a high-fat diet and treated with BA showed less weight gain and tissue adiposity without any change in calorie intake. Gene expression profiling of mouse tissues and cell lines revealed that BA treatment increased expression of lipid oxidative genes and decreased that of lipogenesis-related genes. This modulation was mediated by increased AMP-activated protein kinase (AMPK) phosphorylation, which facilitates energy expenditure, lipid oxidation and thermogenic capacity and exerts protective effects against obesity and nonalcoholic fatty liver disease. Overall, BA markedly inhibited the development of obesity and nonalcoholic fatty liver disease in mice fed a high-fat diet, and AMPK activation in various tissues and enhanced thermogenesis are two possible mechanisms underlying the antiobesity and antisteatogenic effects of BA. CONCLUSIONS: The current findings suggest that treatment with BA is a potential dietary strategy for preventing obesity and nonalcoholic fatty liver disease.

Global transcriptome analysis identifies weight regain-induced activation of adaptive immune responses in white adipose tissue of mice.

OBJECTIVE: Studies have indicated that weight regain following weight loss predisposes obese individuals to metabolic disorders; however, the molecular mechanism of this potential adverse effect of weight regain is not fully understood. Here we investigated global transcriptome changes and the immune response in mouse white adipose tissue caused by weight regain. DESIGN: We established a diet switch protocol to compare the effects of weight regain with those of weight gain without precedent weight loss, weight loss maintenance and chow diet. We conducted a time course analysis of global transcriptome changes in gonadal white adipose tissue (gWAT) during the weight fluctuation. Co-expression network analysis was used to identify functional modules associated with the weigh regain phenotype. Immune cell populations in gWAT were characterized by flow-cytometric immunophenotyping. Metabolic phenotypes were monitored by histological analysis of adipose tissue and liver, and blood-chemistry and body weight/composition analyses. RESULTS: In total, 952 genes were differentially expressed in the gWAT in the weight regain vs the weight gain group. Upregulated genes were associated with immune response and leukocyte activation. Co-expression network analysis showed that genes involved in major histocompatibility complex I and II-mediated antigen presentation and T-cell activation function were upregulated. Consistent with the transcriptome analysis results, flow cytometry demonstrated significant increases in subsets of T cells and proinflammatory M1 macrophages in the gWAT in the weight regain as compared to the weight gain group. In addition, upregulation of adaptive immune responses was associated with high incidence of adipocyte death and upregulation of high mobility group box 1, a well-known component of damage-associated molecular patterns. CONCLUSIONS: Our global transcriptome analysis identified weight regain-induced activation of adaptive immune responses in mouse white adipose tissue. Results suggest that activation of adipocyte death-associated adaptive immunity in adipose tissue may contribute to unfavorable metabolic effects of weight regain following weight loss.

An IF-FISH Approach for Covisualization of Gene Loci and Nuclear Architecture in Fission Yeast.

Recent genomic studies have revealed that chromosomal structures are formed by a hierarchy of organizing processes ranging from gene associations, including interactions among enhancers and promoters, to topologically associating domain formations. Gene associations identified by these studies can be characterized by microscopic analyses. Fission yeast is a model organism, in which gene associations have been broadly mapped across the genome, although many of those associations have not been further examined by cell biological approaches. To address the technically challenging process of the visualization of associating gene loci in the fission yeast nuclei, we provide, in detail, an IF-FISH procedure that allows for covisualizing both gene loci and nuclear structural markers such as the nuclear membrane and nucleolus.

Oxygen-vacancy-induced orbital reconstruction of Ti ions at the interface of LaAlO3/SrTiO3 heterostructures: a resonant soft-X-ray scattering study.

Resonant soft-x-ray scattering measurements have been performed to investigate interface electronic structures of (LaAlO(3)/SrTiO(3)) superlattices. Resonant scattering intensities at superlattice reflections show clear evidence of degeneracy lifting in t(2g) states of interface Ti ions. Polarization dependence of intensities indicates the energy of d(xy) states is lower by ~1 eV than two other t(2g) states. The energy splitting is insensitive to epitaxial strain. The orbital reconstruction is induced by oxygen vacancies and confined to the interface within two unit cells, indicating charge compensation at the polar interfaces.

The volatile-producing Flavobacterium johnsoniae strain GSE09 shows biocontrol activity against Phytophthora capsici in pepper.

AIMS: Previously, we selected a bacterial strain (GSE09) antagonistic to Phytophthora capsici on pepper, which produced a volatile compound (2,4-di-tert-butylphenol), inhibiting the pathogen. In this study, we identified strain GSE09 and characterized some of the biological traits of this strain in relation to its antagonistic properties against P. capsici. In addition, we examined bacterial colonization on the root surface or in rhizosphere soil and the effect of various concentrations of the volatile compound and strain GSE09 on pathogen development and radicle infection as well as radicle growth. METHODS AND RESULTS: Strain GSE09 was identified as Flavobacterium johnsoniae, which forms biofilms and produces indolic compounds and biosurfactant but not hydrogen cyanide (HCN) with little or low levels of antifungal activity and swimming and swarming activities. Fl. johnsoniae GSE09 effectively colonized on pepper root, rhizosphere, and bulk (pot) soil, which reduced the pathogen colonization in the roots and disease severity in the plants. Various concentrations of 2,4-di-tert-butylphenol or strain GSE09 inhibited pathogen development (mycelial growth, sporulation, and zoospore germination) in I-plate (a plastic plate containing a center partition). In addition, germinated seeds treated with the compound (1-100 µg ml⁻¹) or the strain (10²-10¹° cells ml⁻¹) significantly reduced radicle infection by P. capsici without radicle growth inhibition. CONCLUSIONS: These results indicate that colonization of pepper root and rhizosphere by the Fl. johnsoniae strain GSE09, which can form biofilms and produce indolic compounds, biosurfactant, and 2,4-di-tert-butylphenol, might provide effective biocontrol activity against P. capsici. SIGNIFICANCE AND IMPACT OF THE STUDY: To our knowledge, this is the first study demonstrating that the Fl. johnsoniae strain GSE09, as a potential biocontrol agent, can effectively protect pepper plants against P. capsici infection by colonizing the roots.

Tumor necrosis factor-α and interleukin-1ß expression pathway induced by Streptococcus mutans in macrophage cell line RAW 264.7.

Streptococcus mutans, a major etiological agent of dental caries, frequently causes systemic disease, such as subacute bacterial endocarditis, if it enters the bloodstream. In this study, the production pathways of the proinflammatory cytokines, tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß), induced by S. mutans in mouse macrophage were examined using a quantitative real-time polymerase chain reaction and an enzyme-linked immunosorbent assay. The S. mutans stimulated the expression of TNF-α and IL-1ß mRNA at a multiplicity of infection of 1 : 100, which increased at 2 and 4 h, respectively, to 24 h. It also induced the production of high levels of the TNF-α and IL-1ß proteins, which increased at 2 h and reached a peak at 4 and 24 h, respectively. Nuclear factor-κB (NF-κB) was activated and reached a maximum level 30 min after the S. mutans treatment. The expression of TNF-α and IL-1ß mRNA and protein was suppressed by the treatment with pyrrolidine dithiocarbamate, an NF-κB inhibitor. The S. mutans-induced TNF-α expression was suppressed by the presence of SB203580, a p38 mitogen-activated protein (MAP) kinase inhibitor, or SP600125, a Jun N-terminal kinase (JNK) MAP kinase inhibitor. On the other hand, IL-1ß expression was inhibited by extracellular signal-regulated kinase (ERK)/p38/JNK MAP kinase inhibitor pretreatment. In addition, TNF-α production was suppressed more in the Toll-like receptor 2(-/-) (TLR2(-/-)) macrophages than in the TLR4(-/-) macrophages, whereas IL-1ß production was suppressed more in the TLR4(-/-) macrophages than in the TLR2(-/-) macrophages. These results show that S. mutans stimulates the production of TNF-α and IL-1ß in the mouse macrophage cell line, RAW 264.7, by activating ERK/p38/JNK, and NF-κB through TLR2 and TLR4, respectively.

A biometric study of C-shaped root canal systems in mandibular second molars using cone-beam computed tomography.

AIM: To investigate the configuration of C-shaped canals in mandibular second molars, canal wall thickness and the orientation of the thinnest area at 1-mm intervals from the canal orifice to the apex by using cone-beam computed tomographic (CBCT) images. METHODOLOGY: Three-dimensional CBCT images of 92 Korean mandibular second molars having C-shaped root canals were analysed to determine their configuration using a modification of Melton's classification, as well as the thinnest walls and their location. Associations between configuration type and distance from the canal orifice to the apex, as well as associations between the directional orientation of the thinnest root wall and distance from the canal orifice to the apex, were assessed by Fisher's exact test. Because serial measurements of minimum wall thicknesses were correlated with individual teeth, a mixed-effects analysis was applied. RESULTS: The most common configuration types were Melton's type I in the coronal region and Melton's type III in the apical region. Mean thicknesses of the thinnest root canal walls were 1.39 ± 0.38, 0.85 ± 0.25 and 0.77 ± 0.20 mm in the coronal, middle and apical regions, respectively. The thicker the root canal walls at the orifice region, the greater the decrease in thickness towards the apical region (P < 0.05), with the linguo-central root area being the thinnest. The pattern of decreasing thickness from the orifice to the apex formed a nonlinear cubic curve. CONCLUSIONS: The most prevalent configuration types were Melton's type I (coronal region) and type III (apical region). The linguo-central root area was the thinnest in C-shaped root canals of Korean mandibular second molars. These anatomical variations should be considered during surgical or nonsurgical endodontic procedures.

Trabecular bone ratio of the mandibular condyle according to the presence of teeth: a micro-CT study.

PURPOSE: During mastication, mechanical pressure from the dentition is transmitted to the trabecular bone of the mandible. The occlusal forces, which could thus affect condylar growth, vary with tooth loss, age, and sex. The trabecular bone of the mandibular condyle is denser in dentate subjects than in edentate subjects. However, since the different tooth groups (incisor, premolar, and molar) have different functions, they could exert different effects on the mandibular condyle. The aim of this study was to elucidate the bone quantity of the Korean mandibular condyle according to the presence of teeth using micro-computed tomography (micro-CT), thereby clarifying the influences of tooth presence on the condylar microstructure. METHODS: Thirty-one sides were scanned and reconstructed into a 3D structure using a micro-CT system. The specimen was sectioned vertically, passing through the medial and lateral poles of the mandibular condyle (P0) to enable measurement of the trabecular bone ratio. Likewise, three additional images, parallel with P0, were acquired. Mean and standard deviation values were calculated, and the t test, one-way ANOVA and post hoc analysis were performed to examine the differences among each group, classified according to the presence of teeth and according to sex. RESULTS: The density of the trabecular bone of the mandibular condyle was significantly associated with the presence of the molars, but not the incisors or premolars. There were significant differences between sexes. CONCLUSIONS: The present study has provided data regarding the bone quantity of the trabeculae of the mandibular condyle according to the presence or absence of teeth.

Autotransplantation of an ectopic impacted premolar with sinus lift and allogenic bone graft.

AIM: This article presents a case report of autogenous tooth transplantation to a site which had insufficient bone volume using a sinus lift and allogenic bone graft. SUMMARY: An ectopic, fully impacted premolar tooth was autotransplanted from its ectopic impaction site into its original site, where there was insufficient recipient vertical bone volume because of sinus expansion. The deciduous tooth was extracted from the recipient site, and the sinus membrane detached and elevated via the alveolar socket. Allogenic bone grafting was performed, and the impacted premolar was transplanted into the prepared site. To improve adaptation, the recipient site was prepared using a rapid prototype tooth model, a replica tooth which allowed complete socket preparation in advanced of the actual removal of the donor tooth. The donor tooth was fixed with sutures and maintained for 17 days to allow physiologic movement. Root canal treatment was initiated 24 days after autotransplantation, and an intra-canal medicament was used for 4 months. Canal filling was completed 5 months after autotransplantation. There was no root resorption of the transplanted tooth, and the grafted bone was well preserved and had no signs of infection. KEY LEARNING POINTS: When the recipient bone volume is insufficient, autotransplantation can be preceded by bony augmentation. The preparation of the recipient tooth socket using a tooth replica from CBCT reduces the extra-oral time of the actual tooth and promotes better periodontal ligament healing. Careful evaluation of the pulp status of the donor tooth is important in advance of timely endodontic treatment.

Allelic variation of the Waxy gene in foxtail millet [Setaria italica (L.) P. Beauv.] by single nucleotide polymorphisms.

The Waxy (Wx) gene product controls the formation of a straight chain polymer of amylose in the starch pathway. Dominance/recessiveness of the Wx allele is associated with amylose content, leading to non-waxy/waxy phenotypes. For a total of 113 foxtail millet accessions, agronomic traits and the molecular differences of the Wx gene were surveyed to evaluate genetic diversities. Molecular types were associated with phenotypes determined by four specific primer sets (non-waxy, Type I; low amylose, Type VI; waxy, Type IV or V). Additionally, the insertion of transposable element in waxy was confirmed by ex1/TSI2R, TSI2F/ex2, ex2int2/TSI7R and TSI7F/ex4r. Seventeen single nucleotide polymorphims (SNPs) were observed from non-coding regions, while three SNPs from coding regions were non-synonymous. Interestingly, the phenotype of No. 88 was still non-waxy, although seven nucleotides (AATTGGT) insertion at 2,993 bp led to 78 amino acids shorter. The rapid decline of r (2) in the sequenced region (exon 1-intron 1-exon 2) suggested a low level of linkage disequilibrium and limited haplotype structure. K (s) values and estimation of evolutionary events indicate early divergence of S. italica among cereal crops. This study suggested the Wx gene was one of the targets in the selection process during domestication.

Reduction of metal artifact in three-dimensional computed tomography (3D CT) with dental impression materials.

Metal Artifact still remains one of the main drawbacks in craniofacial Three-Dimensional Computed Tomography (3D CT). In this study, we tried to test the efficacy of additional silicone dental impression materials as a "tooth shield" for the reduction of metal artifact caused by metal restorations and orthodontic appliances. 6 phantoms with 4 teeth were prepared for this in vitro study. Orthodontic bracket, bands and amalgam restorations were placed in each tooth to reproduce various intraoral conditions. Standardized silicone shields were fabricated and placed around the teeth. CT image acquisition was performed with and without silicone shields. Maximum value, mean, and standard deviation of Hounsfield Units (HU) were compared with the presence of silicone shields. In every situation, metal artifacts were reduced in quality and quantity when silicone shields are used. Amalgam restoration made most serious metal artifact. Silicone shields made by dental impression material might be effective way to reduce the metal artifact caused by dental restoration and orthodontic appliances. This will help more excellent 3D image from 3D CT in craniofacial area.

Fabrication of fluorine-doped diamond-like carbon stamps for UV nanoimprint lithography.

Two fluorine-doped diamond-like carbon (F-DLC) stamps with sub-100 nm line patterns were fabricated using a direct etching method. These were applied successfully to ultraviolet (UV) nanoimprint lithography without requiring an anti-adhesion layer coating. Tests were performed to determine the optimum fluorine concentration for the F-DLC stamps. The ideal stamp material consisted of 25 at.% F-DLC with a contact angle of 85°, UV transmittance of 16.4-26.8%, and hardness of 4.5 GPa. The O(2) plasma etch rate of the DLC was increased considerably by the fluorine doping, making it comparable to the etch rate of polymethyl methacrylate (PMMA). Thus, only PMMA was used as the etch mask in the fabrication of the stamps.

Buccal bone plate thickness of the Asian people.

Distances from the apex to the buccal bone plate were measured on the computed tomography (CT) images of 1806 teeth from 66 patients, using an image analyzer program (Image-Pro Plus, Ver. 4.0, Media Cybernetics). In the mandible, the mean distance from the distal apex of the mandibular second molar to the buccal bone plate was the largest distance measured, at 8.51 mm, followed by distance from the mesial root to the buccal bone (7.34 mm). In the mandibular first molar, the mean distal and mesial bone thicknesses were 5.18 mm and 4.09 mm, respectively. However, when there were two distal roots, the distance of the disto-lingual root to the buccal plate was found to be 9.52 mm, which constitutes the greatest measured thickness. In the maxillary buccal roots, the distances from the mesio-buccal and disto-buccal root of the second molar to the buccal bone plate were the largest, at 4.63 mm and 3.61 mm, respectively. The average distances from the palatal apex of the maxillary first and second molars to the buccal bone plate were 10.69 mm and 10.17 mm, respectively, while, from the palatal bone plate, average distances of 3.15 mm and 3.08 mm were measured. Special considerations, such as bony lid approach, lingual approach, or intentional replantation may be required, especially when a patient has a surgical need in the second molars and the disto-lingual root of the mandibular first molar, or in the palatal root of the maxillary molars.

The three-dimensional microstructure of the trabecular bone in the mandible.

This study investigated the three dimensional (3D) trabecular microstructure of the alveolar and basal bone in the mandible using micro-CT and compared the morphometric values of the different sites. Ten specimens were prepared and scanned using a micro-CT system. Both the alveolar and basal trabecular bone of the premolar region in the mandible were measured for the structural analysis. Cross-sectional 1024x1024 pixel images were created. From the two-dimensional (2D) images produced, 3D structural images were reconstructed. After scanning the specimen, the volumes of interest (VOI) of the alveolar and basal bone regions were selected from the 3D reconstruction images, and the structural parameters such as bone volume fraction, bone surface density, trabecular thickness, trabecular separation, trabecular number and structural model index were analyzed. The trabecular structure showed a marked variation within the sites of the specimen, especially in the basal trabecular bone inferior to the mandibular canal. In both the alveolar and basal bone regions, a mixture of both plate-like and rod-like structures was observed. The alveolar region showed a more compact, plate-type trabecular structure than the basal regions. In parametric comparison with the basal bone, the alveolar bone generally had a higher bone volume fraction, bone trabecular thickness and trabecular number, and lower bone surface density, trabecular separation and structural model index. The alveolar bone consisted of a compact bone structure with a large amount of thick plate-type trabecular bone, which was effectively resistant to the masticatory forces. As the measurements were made closer to the basal bone, a loose structure was observed with lower bone volume and fewer, thin, rod-like trabeculae.

Personal-computer-based three-dimensional reconstruction and simulation of maxillary sinus.

Anatomical descriptions of the maxillary sinus are critical in pathological diagnosis and the treatment planning of surgical procedures. This study was undertaken to develop a new technique for simulating anatomical structures and to clarify the morphological and clinical characteristics of the maxillary sinus. Thirty-three hemi-sectioned Korean heads were used in this study. CT scans and DentaScan reformatted cross-sectional images were taken on all specimens. From the CT images, three-dimensional reconstructed images were made using the V-works program. From the three-dimensional reconstructed images of the maxillary sinus, six categories of maxillary sinus were created, categorized according to their lateral aspects and shapes of the inferior walls. In 55%, a flat inferior wall of the maxillary sinus was observed. All measurements (anterior-posterior length, height, width and volume) of the sinus were larger in males than in females. From the DentaScan reformatted panoramic images, the anterior limit of the maxillary sinus was located in the first premolar area (58%), and the posterior limit was in the third molar and maxillary tuberosity area (94%). We therefore offer a new virtual technique for manipulating three-dimensional reconstructed images easily on a personal computer. On the reconstructed images the three-dimensional morphology could be observed and the anatomical characteristics of the maxillary sinus and surrounding structures could be determined.

Inflammatory orbital pseudotumor with infratemporal fossa extension mimicking temporomandibular joint dysfunction.

Inflammatory orbital pseudotumor is a benign space-occupying lesion of unknown origin that involves all or part of the fatty tissue within the orbit. Occasionally the disease may extend into the middle cranial, the pterygopalatine, and the infratemporal fossa through the various foramina of the orbit, although extension into the infratemporal fossa is very rare. We present a case which extends into the infratemporal fossa through the inferior orbital fissure, resulting in presenting symptoms mimicking temporomandibular joint dysfunction.

Localization of peripheral dopamine D1 and D2 receptors in rat corpus cavernosum.

OBJECTIVE: To detect and locate anatomically peripheral dopamine D1 and D2 receptors in rat cavernosa, as dopamine is important in sexual drive and penile erection through receptors located in the central nervous system. MATERIALS AND METHODS: Corpora cavernosa were obtained from Sprague-Dawley rats; total RNA and membrane proteins were extracted and cryostat sections prepared. The rat brain hypothalamus was used as a control for dopamine D1 and D2 receptors. The presence and expression of peripheral dopamine D1 and D2 receptor mRNAs in rat corpus cavernosa was assessed using reverse transcription and polymerase chain reaction (RT-PCR), and Northern blot hybridization using (32)P-UTP-labelled RNA probes. Concurrently, corresponding proteins from D1 and D2 receptors were assayed and detected by a Western blotting technique. The anatomical location of dopamine D1 and D2 receptor mRNAs in rat penile tissues was identified by in situ hybridization using (35)S-UTP-labelled RNA probes in cryostat sections. Immunohistochemical staining was used to locate peripheral dopamine D1 and D2 receptor proteins in rat corpora cavernosa. RESULTS: Dopamine D1 and D2 receptor gene expression was detected in rat corpora cavernosa. In situ hybridization signals for dopamine D1 and D2 receptor mRNAs were localized to corpus cavernosal tissues and dorsal vessels in the rat penis. Western blot analyses showed peripheral dopamine D1 and D2 receptor proteins in rat corpora cavernosa. Immunohistochemically, peripheral dopamine D1 and D2 receptor proteins were detected in dorsal nerves, dorsal vessels and corpus cavernosal smooth muscle of the rat penile tissues. CONCLUSIONS: Peripheral dopamine D1 and D2 receptors are present in the corpora cavernosa of rats. The functional significance of these receptors and signal transduction pathways in modulating the vascular tone of the penis warrants further investigation.

Knowledge and attitudes of Korean parents towards their son's circumcision: a nationwide questionnaire study.

OBJECTIVE: To evaluate knowledge about the foreskin and circumcision, and to understand the attitudes of parents to circumcision in Korea, where circumcision in childhood is widely practised with no particular religious or medical background. SUBJECTS AND METHODS: A nationwide study involving questionnaires was conducted on 5500 parents with at least one son attending elementary school. Responses were obtained from one of the parents. RESULTS: The response rate was 76.1% (4183); circumcision was most common in boys when aged 11 years, followed by neonatal circumcision. Of the parents, 91.3% believed that circumcision is necessary, while 2.1% believed it to be unnecessary. The principal reasons given for circumcision were 'to improve penile hygiene' (82.4%), followed by 'to improve future sexual potency' (7.5%). Among those who did not believe circumcision to be necessary, the most common reason was the expectation of spontaneous retraction of the prepuce with age (55.1%). Most (88.4%) of the parents believed that smegma is not a clean material, and is infected by microorganisms. Most parents (80.6%) thought that circumcision would prevent genital tract infection of the future spouse. Peer pressure was one of the most influential factors in deciding upon circumcision; 41.9% of the parents were anxious that their child might be ridiculed by his peer group unless he was circumcised, while 27.4% of the parents believed that their child might be ridiculed if he was circumcised. Mothers were more positive about circumcision than fathers (P < 0.05). Parents with a higher education and higher socio-economic status were also more positive about circumcision (P < 0.05). Mothers were prone to emphasize improved sexual potency (P < 0.05). There was no significant difference in response between urban and rural areas. CONCLUSION: This study indicates that common beliefs held by parents about the prepuce or circumcision differ significantly from current medical knowledge, and these beliefs have a major influence on the practice of circumcision in Korea. More clinical research on the natural history of the foreskin is needed, and it is critical that both children and parents are informed about the potential benefits and disadvantages of circumcision.

Isolation of neuronal precursors from differentiating P19 embryonal carcinoma cells by neuronal T alpha 1-promoter-driven GFP.

The induction of pluripotent P19 embryonal carcinoma (EC) cells with retinoic acid results in their differentiation into cells that resemble neurons, glia, and fibroblasts. To isolate and enrich the developing neurons from heterogeneously differentiating P19 EC cells, we used a recently introduced protocol combining the expression of green fluorescent protein (GFP) driven by a tissue-specific promoter and fluorescence-activated cell sorting. Cells were transfected with the gene for GFP, which is under the control of the neuronal T alpha 1 tubulin promoter. After four days of retinoic acid treatment, GFP was specifically detected in cells undergoing neuronal differentiation. Sorting of fluorescent differentiating P19 EC transfectants yielded populations highly enriched in neuronal precursors and neurons. Immunoreactivity for nestin and neurofilament was observed in 80 and 25% of the sorted cell population, respectively. These results demonstrate that differentiated neuronal precursor cells can be efficiently isolated from differentiating pluripotent embryonic cells in vitro, suggesting that this method can reproducibly provide homogeneous materials for further studies on neurogenesis.