Skin autofluorescence in people with type 1 diabetes and people without diabetes: An eight-decade cross-sectional study with evidence of accelerated aging and associations with complications.

AIM: To measure skin autofluorescence in youth (<18 y.o.) and adults (≥18 y.o.) and to assess its relationship with type 1 diabetes, chronic complications and smoking. METHODS: In a cross-sectional study (n = 383) skin autofluorescence was measured in 269 people with type 1 diabetes (67 with vascular complications) and 114 people without diabetes, covering eight decades of age. Associations of skin autofluorescence with demographics and traditional risk factors were assessed. RESULTS: Skin autofluorescence increased with age in people with diabetes: for those with complications it increased by a mean ± se of 0.029 ± 0.003 arbitrary units per year (r = 0.76) and, for those without complications, it increased by 0.028 ± 0.002 arbitrary units (r = 0.77). These increases were higher than for people without diabetes, whose skin autofluorescence increased by 0.022 ± 0.002 arbitrary units (r = 0.78) per year (p = 0.004). Mean ±se age-adjusted skin autofluorescence was higher in people with diabetes complications vs people without diabetes complications (1.85 ± 0.04 vs 1.66 ± 0.02 arbitrary units) and people without diabetes (1.48 ± 0.03 arbitrary units; all P < 0.0001). Age-adjusted skin autofluorescence was higher in current smokers and recent ex-smokers vs non-smokers and longer-term ex-smokers (1.86 ± 0.06 vs 1.63 ± 0.02 arbitrary units; P = 0.0005). Skin autofluorescence area under the receiver-operating characteristic curve was 0.89 (95% CI 0.85-0.94) for retinopathy and 0.56 (95% CI 0.47-0.65) for nephropathy. CONCLUSIONS: Skin autofluorescence increases with age, but faster in people with diabetes, particularly in those with complications and in smokers, consistent with accelerated aging. Skin autofluorescence may facilitate complication screening and prediction. Longitudinal studies are merited.

Early changes of arterial elasticity in Type 1 diabetes with microvascular complications - A cross-sectional study from childhood to adulthood.

AIM: To examine the trajectory of small artery elasticity (SAE) and pulse pressure (PP) in people with Type 1 diabetes and non-diabetic controls across the lifespan, and explore associations with microvascular complications (CX+). METHODS: This cross-sectional study included 477 Type 1 diabetes patients (188 with CX+, 289 without CX-) and 515 controls. Relationships between SAE and PP and age were evaluated using segmented linear regression. Logistic regression was used to assess the associations between microvascular complications (retinopathy and/or nephropathy) and SAE and PP. RESULTS: SAE peaked significantly later among controls than diabetic patients CX- vs. CX+ (21.2 vs. 20.4 vs. 17.6 years respectively, p < 0.001). In adults, mean SAE was significantly lower in CX+ vs. CX- vs. controls (6.8 vs. 7.8 vs. 8.0 ml/mm Hg × 10; p < 0.0001), and mean PP was significantly higher in CX+ vs CX- and controls (60 vs. 55 vs. 53 mm Hg; p < 0.0001). CONCLUSION: Type 1 diabetes CX+ subjects have an earlier peak and decline in SAE relative to CX- and controls, who did not differ. Lower SAE and higher PP were associated with increased odds of Type 1 diabetes complications in adults. These clinically applicable techniques demonstrate an association between accelerated vascular aging and vascular complications in diabetes.

Activation of nuclear PTEN by inhibition of Notch signaling induces G2/M cell cycle arrest in gastric cancer.

Mutation in PTEN has not yet been detected, but its function as a tumor suppressor is inactivated in many cancers. In this study we determined that, activated Notch signaling disables PTEN by phosphorylation and thereby contributes to gastric tumorigenesis. Notch inhibition by small interfering RNA or γ-secretase inhibitor (GSI) induced mitotic arrest and apoptosis in gastric cancer cells. Notch inhibition induced dephosphorylation in the C-terminal domain of PTEN, which led to PTEN nuclear localization. Overexpression of activated Notch1-induced phosphorylation of PTEN and reversed GSI-induced mitotic arrest. Dephosphorylated nuclear PTEN caused prometaphase arrest by interaction with the cyclin B1-CDK1 complex, resulting in their accumulation in the nucleus and subsequent apoptosis. We found a correlation between high expression levels of Notch1 and low survival rates and, similarly, between reduced nuclear PTEN expression and increasing the TNM classification of malignant tumours stages in malignant tissues from gastric cancer patients. The growth of Notch1-depleted gastric tumors was significantly retarded in xenografted mice, and in addition, PTEN deletion restored growth similar to control tumors. We also demonstrated that combination treatment with GSI and chemotherapeutic agents significantly reduced the orthotopically transplanted gastric tumors in mice without noticeable toxicity. Overall, our findings suggest that inhibition of Notch signaling can be employed as a PTEN activator, making it a potential target for gastric cancer therapy.

Short-term effect of gastric resection on circulating levels of ghrelin, peptide YY3-36 and obestatin in patients with early gastric cancer.

The short-term responses of gut hormones and the compensative interaction during a one-week period after subtotal gastrectomy in early gastric cancer (EGC) patients were assessed. Previous studies have reported gut hormonal changes after Roux-en-Y gastric bypass surgery. Blood samples were collected from 40 patients with EGC preoperatively, at 1 h after gastric resection, and on postoperative day (POD) 1, 3, and 7. Levels of active ghrelin, total ghrelin, obestatin, and PYY3-36 were measured. Total ghrelin level rapidly reached a nadir of 69.1%, while active ghrelin level had increased to 135.5% at 1 h after resection. Then, both returned to preoperative level. On the contrary, active/total ghrelin reached its nadir quickly at 1 h after resection and had returned to the preoperative level by POD 3. The nadir PYY3-36 level was 71.4% on POD 1, followed by a gradual recovery, and had increased to 116.5% by POD 7. The same pattern was observed for obestatin. Active ghrelin/obestatin showed an increase on POD 1 while total ghrelin/obestatin showed a decrease on POD 3. Then, both returned to preoperative level. These results suggest that a rapid interactive compensatory mechanism of gut hormones does exist in the remnant gastrointestinal tract after abrupt changes in the production reservoir in nonobese people.

Clinical and molecular characterization of a novel PLIN1 frameshift mutation identified in patients with familial partial lipodystrophy.

Perilipin 1 is a lipid droplet coat protein predominantly expressed in adipocytes, where it inhibits basal and facilitates stimulated lipolysis. Loss-of-function mutations in the PLIN1 gene were recently reported in patients with a novel subtype of familial partial lipodystrophy, designated as FPLD4. We now report the identification and characterization of a novel heterozygous frameshift mutation affecting the carboxy-terminus (439fs) of perilipin 1 in two unrelated families. The mutation cosegregated with a similar phenotype including partial lipodystrophy, severe insulin resistance and type 2 diabetes, extreme hypertriglyceridemia, and nonalcoholic fatty liver disease in both families. Poor metabolic control despite maximal medical therapy prompted two patients to undergo bariatric surgery, with remarkably beneficial consequences. Functional studies indicated that expression levels of the mutant protein were lower than wild-type protein, and in stably transfected preadipocytes the mutant protein was associated with smaller lipid droplets. Interestingly, unlike the previously reported 398 and 404 frameshift mutants, this variant binds and stabilizes ABHD5 expression but still fails to inhibit basal lipolysis as effectively as wild-type perilipin 1. Collectively, these findings highlight the physiological need for exquisite regulation of neutral lipid storage within adipocyte lipid droplets, as well as the possible metabolic benefits of bariatric surgery in this serious disease.

Aerosol delivery of eukaryotic translation initiation factor 4E-binding protein 1 effectively suppresses lung tumorigenesis in K-rasLA1 mice.

Conventional radiotherapy or chemotherapy for the long-term survival of patients with lung cancer is still difficult for treatment in metastatic and advanced tumors. Therefore, the safe and effective approaches to the treatment of lung cancer are needed. In this study, the effect of delivered eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) on lung cancer progression was evaluated. Recombinant adeno-associated virus (rAAV)-M3/4E-BP1 was delivered into 6-week-old K-rasLA1 lung cancer model mice through a nose-only inhalation system twice a week for 4 weeks. Long-term repeated delivery of 4E-BP1 effectively reduced tumor progression in the lungs of K-rasLA1 mice. Reduction of eIF4E by overexpression of 4E-BP1 resulted in suppression of cap-dependent protein expression of basic fibroblast growth factor (bFGF or FGF-2) and vascular endothelial growth factor (VEGF). In addition, delivered 4E-BP1 inhibited the proliferation of lung cancer cells in K-rasLA1 mice model. Our results suggest that long-term repeated viral delivery of 4E-BP1 may provide a useful tool for designing lung cancer treatment.

Melanocyte-specific CD8+ T cells are associated with epidermal depigmentation in a novel mouse model of vitiligo.

In the present study, we established a novel murine model of vitiligo by sequential prime/boost immunizations into the hind footpad and tail dermis with tyrosinase-related protein 2 (TRP2)-180 (SVYDFFVWL) peptide, lipopolysaccharides and cytosine-phosphate-guanosine (CpG) oligodeoxynucleotides. Immunized mice developed epidermal depigmentation in the tail skin without hair depigmentation, thereby differentiating this approach from established models of vitiligo. Following intradermal tail immunization, activated CD8(+) interferon (IFN)-γ(+) T cells were recruited locally to the tail skin. In-vivo cytotoxicity assays demonstrated specific lysis of TRP2-180-presenting cells in immunized mice. Furthermore, the extent of skin depigmentation correlated with the frequency of TRP2-180-specific splenic CD8(+) T cells, as determined by IFN-γ and tumour necrosis factor (TNF)-α production, and cytotoxic degranulation evidenced by CD107a staining. These findings suggest a correlation between the presence of TRP2-180-specific CD8(+) effector T cells and the development of depigmented skin lesions in our vitiligo model. This new model of vitiligo, characterized by skin depigmentation without hair depigmentation, is more similar to human disease than previous murine models. Therefore, this model is well suited to future studies on the pathogenesis of vitiligo and the development of novel therapeutics for vitiligo.

Cleavage of BCR-ABL transcripts at the T315I point mutation by DNAzyme promotes apoptotic cell death in imatinib-resistant BCR-ABL leukemic cells.

The BCR-ABL fusion transcript encodes the BCR-ABL tyrosine kinase (TK), which causes chronic myelogenous leukemia (CML). Although the TK inhibitor imatinib mesylate, which targets the BCR-ABL protein, has been proven to be effective in controlling leukemic growth, imatinib resistance has been observed with disease relapse because of point mutations in the ABL gene that inhibit imatinib efficacy. In this study, we designed oligodeoxyribozymes (DNAzymes) that specifically target and cleave both the junction sequence and the site of the point mutation (T315I), conferring imatinib resistance in BCR-ABL mRNA. DNAzymes significantly induced apoptosis and inhibited proliferation in wild-type and T315I-mutant BCR-ABL-positive cells. Selective cleavage of T315I-mutant ABL mRNA by DNAzyme (T315I Dz) led to cell cycle arrest in G0/G1 phase, with induction of caspase-3/-7 in imatinib-resistant BCR-ABL-positive cells harboring the T315I mutation. Moreover, cotreatment with the DNAzyme targeting the T315I mutation and imatinib resulted in enhanced inhibition of proliferation and induction of apoptosis in T315I leukemic cells as compared with imatinib alone, thereby antagonizing imatinib resistance in CML cells bearing T315I-mutant BCR-ABL. Therefore, cleavage of T315I-mutant ABL mRNA by DNAzyme combined with imatinib treatment may be an alternative approach to overcoming imatinib resistance in leukemic cells.

Nonmuscle myosin heavy chain and histone H3 are intracellular binding partners of lithospermic acid B and mediate its antiproliferative effect on VSMCs.

Lithospermic acid B (LAB), an active component of danshen, is known to inhibit the proliferation of vascular smooth muscle cells (VSMCs) and has pharmacological activity scavenging free radicals in VSMCs. However, the precise mechanism through which LAB exerts its antiproliferative effect is unclear. Therefore, we investigated how LAB regulates cellular proliferation in primary cultured rat VSMCs. Using fluorescein isothiocyanate (FITC)-conjugated LAB to track its cellular localization, we show that LAB localizes to the nucleus, specifically to the nucleolus, where it binds to histone H3, leading to the inhibition of the platelet-derived growth factor (PDGF)- induced phosphorylation of histone H3. LAB also only moves into the nucleus during the normal expression of nonmuscle myosin heavy chain (NMHC-IIA), which is associated with LAB in VSMCs. Notably, LAB suppressed the PDGF-induced phosphorylation of Akt and the expression of cyclin D2 in the presence of NMHC-IIA expression. Knockdown of NMHC-IIA expression impeded the function of LAB, which was then unable to inhibit the PDGF-induced proliferation of VSMCs. We conclude that LAB modulates the PDGF-induced proliferation of VSMCs by interacting with NMHC-IIA, which allows LAB to localize in the nucleus and to suppress the PDGF-induced proliferation of VSMCs.

Smad3 regulates E-cadherin via miRNA-200 pathway.

To identify potential microRNA (miRNA) links between Smad3, a mediator of TGF-ß (transforming growth factor-ß) signaling, and E-cadherin, we characterized the miRNA profiles of two gastric cancer cell lines: SNU484-LPCX, which does not express Smad3, and SNU484-Smad3, in which Smad3 is overexpressed. We found that among differentially expressed miRNAs, miR-200 family members are overexpressed in SNU484-Smad3 cells. Subsequent studies, including analysis of the effects of silencing Smad3 in SNU484-Smad3 cells and a luciferase reporter assay, revealed that Smad3 directly binds to a Smad-binding element located in the promoter region of miR-200b/a, where it functions as a transcriptional activator. TGF-ß did not affect the regulatory role of Smad3 in transcription of miR-200 and expression of epithelial-mesenchymal transition markers. We conclude that Smad3 regulates, at the transcriptional level, miR-200 family members, which themselves regulate ZEB1 and ZEB2, known transcriptional repressors of E-cadherin, at the posttranscriptional level in a TGF-ß-independent manner. This represents a novel link between Smad3 and posttranscriptional regulation by miRNAs in epithelial-mesenchymal transition in gastric cancer cells.

Development of an effective method for dendritic cell immunotherapy of mouse melanoma.

Dendritic cell (DC) immunotherapy is a strong candidate for the treatment of incurable cancers especially malignant melanoma. Nevertheless, the proper guideline of DC immunotherapy does not exist. The absence of the guideline is also an obstacle to clinical trials of DC immunotherapy. So we conducted this study in order to develop an effective DC preparation method for immunotherapy in mouse malignant melanoma. Mouse bone marrow-derived DC were stimulated with tumour antigen alone or tumour antigen plus a cocktail (anti-CD40 antibody +TNF-alpha+ IL-1beta) for 8, 24 or 48 h and the characteristics of these DC, such as surface molecules (CD40, CD80, CD86, MHC class II, CCR7), cytokines(IL-12, IFN-gamma, and IL-10), DC-induced T cell proliferation in vitro, and the production of IFN-gamma by those cells, were evaluated. Mice with melanoma were then treated with DC stimulated with tumour antigen alone and tumour antigen plus cocktail for 8 or 48 h. The tumour size and survival rate of these mice were then evaluated. (1) Beneficial clinical effects such as a reduction of tumour size and an increased survival rate were best observed in the group treated with DC stimulated for 8 h with tumour antigen plus cocktail. (2) The single prominent characteristic of DC stimulated for 8 h with tumour antigen plus cocktail was an elevated IL-12 secretion. The cytokine IL-12 was not secreted by other DC. Consequently, proper production of IL-12 was found to be an important requirement for DC used in immunotherapy of mouse melanoma.

CEACAM5 and CEACAM6 are major target genes for Smad3-mediated TGF-beta signaling.

The carcinoembryonic antigen (CEAs) family consists of a large group of evolutionarily and structurally divergent glycoproteins. The transforming growth factor-beta (TGF-beta) signaling pathway has been implicated in the stimulation of CEA secretion in TGF-beta-sensitive colon cells, thereby possibly modulating cell adhesion and differentiation. However, the specific CEAs targeted by TGF-beta signaling or underlying mechanism of the expression of CEAs has not yet been clarified. In this study, we investigated the specific CEAs targeted by the TGF-beta signaling pathway. In nine human gastric cancer cell lines examined, TGF-beta-responsive cell lines showed positive expression of CEAs. Expression patterns of CEA proteins correlated well with the level of CEA (CEACAM5) and CEACAM6 transcripts in these cell lines, but CEACAM1 expression was not observed in all of these cells. To investigate the role of TGF-beta signaling in CEA expression, we selected two TGF-beta unresponsive gastric cancer cell lines; SNU638 cells that contain a mutation in the TGF-beta type II receptor and SNU484 cells that express low to undetectable level of the TGF-beta pathway intermediate protein, Smad3. Restoration of TGF-beta signaling in these cells induced expression of the CEAs and increased activity of both CEA (CEACAM5) and CEACAM6 promoters. CEA expression was observed in the epithelium of the stomach of wild-type mice, but was markedly decreased in Smad3 null mice. These findings suggest that CEA (CEACAM5) and CEACAM6 are major target genes for Smad3-mediated TGF-beta signaling.

New cosmetic agents for skin whitening from Angelica dahurica.

To develop a new whitening agent for cosmetics from natural products, Angelica dahurica was selected for its inhibitory effect on melanogenesis in B16 melanoma cells. From the mechanism study, it was clarified that the ethanolic extracts of this plant showed the suppression of tyrosinase synthesis but no inhibition of tyrosinase activity. In order to find the active constituents from this plant, the ethanol extracts were chromatographed repeatedly with silica gel. Two coumarin compounds were isolated from A. dahurica. Their structures were identified by physicochemical and spectral data such as UV, IR, NMR, and MS. It was shown that the active substance was isoimperatorin (10-[(3-methyl-2-butenyl)oxy]-7H-furo[3,2-g][1] benzopyran-7-one) and imperatorin (9-[(3-methyl-2-butenyl)oxy]-7H-furo[3,2-g][1] benzopyran-7-one). They significantly inhibited tyrosinase synthesis in B16 melanoma cells. To elucidate the action mechanism of the active compounds of A. dahurica, we investigated the changes in the mRNA level of tyrosinase using the RT-PCR technique. As a result, the mRNA level of tyrosinase was markedly reduced by active compounds of A. dahurica. From these results, we suggest that these extracts might be useful as a new whitening agent in cosmetics, but the in vitro findings must be verified in in vivo skin-lightening studies.

The behavioral effect of human mesenchymal stem cell transplantation in cold brain injured rats.

We investigated the effect of stereotaxically transplanted human mesenchymal stem cells (hMSCs) on behavioral change after traumatic cold brain injury in adult rats. Cortical lesions (n= 20) were induced by touching a metal stamp, cooled with liquid nitrogen, to the dura over the forelimb motor cortex of adult rats. The procedure produced a localized lesion, and the animals showed significant motor deficits. hMSCs were freshly isolated from human iliac bone and cultured in tissue culture flasks with 10 ml Dulbecco's modified Eagle's medium. The animals received hMSC grafts (3 x 10(5) hMSCs) 6 days after cold lesion (n = 10). All rats were sacrificed 3 or 7 weeks after cold injury, and immunohistochemical staining was performed on brain sections to identify donor hMSCs. Neurological evaluations were performed with the forepaw adjusting step test and modified neurological scoring. Treatment with 3 x 10(5) hMSCs improved the rat's neurological functions. We also found that the transplanted cells successfully migrated into the injured brain, preferentially localized around the injury site, and expressed the neuronal and astrocyte marker. These data suggest that hMSCs may be a potential therapeutic tool for brain injuries.

Estimated daily intakes of butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and tert-butyl hydroquinone (TBHQ) antioxidants in Korea.

The study was conducted to establish the estimated daily intake (EDI) of antioxidants such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and tert-butyl hydroquinone (TBHQ) in Korea. The EDIs were obtained from two sources. One of the estimations was based on the analytical determination of BHA, BHT and TBHQ in 12 food categories (ten food categories for TBHQ) and on individual dietary intake data obtained from the National Health and Nutrition Survey in 1998 (n=11 525, age > 1 year). The other EDIs of BHA, BHT and TBHQ were based on the maximum permitted levels specified in national food standards in Korea and on individual dietary intake data obtained from the National Health and Nutrition Survey in 1998 (n=11 525, age > 1 year). To establish the EDIs based on the analytical determination and on individual dietary intake data, 133 food samples in 12 food categories were selected from the foods considered to be representative sources of BHA, BHT and TBHQ in the Korean diet. Selected samples were analysed by GC with FID. BHA was not detected in any of the samples analysed. BHT and TBHQ were detected in the samples, but the levels were significantly lower than their maximum limits. The EDIs1 of BHT, and TBHQ for average consumers were 0.0156(-3), and 0.0012(-3) mg kg(-1) body weight bw day(-1) and as a proportion of the ADI were 0.0052 and 0.0002%, respectively. For 95th percentile consumers, the EDIs of BHT and TBHQ were 0.0080 and 0.0006 mg kg(-1) bw day(-1), and as a proportion of the ADI were 2.67 and 0.09%, respectively. EDIs for BHA, BHT and TBHQ based on the maximum permitted levels and on individual dietary intake data were 0.04, 0.04 and 0.04 mg kg(-1) bw day(-1), respectively. The EDIs of BHA, BHT and TBHQ for average consumers ranged from 6.00 to 14.42% of the ADI of each antioxidant. According to these results, the EDIs of BHA, BHT and TBHQ in Korea were significantly lower than ADI of these antioxidants established by the JECFA.

Enhanced expression of glutamate decarboxylase 65 improves symptoms of rat parkinsonian models.

In this study, we report the amelioration of parkinsonian symptoms in rat Parkinson's disease (PD) models, as a result of the expression of glutamate decarboxylase (GAD) 65 with a modified cytomegalovirus (CMV) promoter. The transfer of the gene for gamma-amino butryic acid (GAD), the rate-limiting enzyme in gama-amino butrylic acid (GABA) production, has been investigated as a means to increase inhibitory synaptic activity. Electrophysiological evidence suggests that the transfer of the GAD65 gene to the subthalamic nucleus (STN) can change the excitatory output of this nucleus to inhibitory output. Our in vitro results also demonstrated higher GAD65 expression in cells transfected with the JDK promoter, as compared to cells transfected with the CMV promoter. Also, a rat PD model in which recombinant adeno-associated virus-2 (rAAV2)-JDK-GAD65 was delivered into the STN exhibited significant behavioral improvements, as compared to the saline-injected group. Interestingly, we observed that these behavioral improvements were more obvious in rat PD models in which rAAV2-JDK-GAD65 was injected into the STN than in rat PD models in which rAAV2-CMV-GAD65 was injected into the STN. Moreover, according to electrophysiological data, the rAAV2-JDK-GAD65-injected group exhibited more constant improvements in firing rates than did the rAAV2-CMV-GAD65-injected group. These data indicate that the JDK promoter, when coupled with GAD65 expression, is more effective with regard to parkinsonian symptoms than is the CMV promoter.

Host cell proteins binding to the encapsidation signal epsilon in hepatitis B virus RNA.

The highly conserved encapsidation signal (epsilon) of hepatitis B viral (HBV) pregenomic RNA has been reported as an essential component for encapsidation and protein priming of HBV polymerase. Here, we report that two HBV epsilon RNA-binding host proteins (80 and 43 kDa) and a copurifying protein (100 kDa) were purified and characterized by the combined methods of UV cross-linking analysis with the epsilon RNA and column chromatography. Amino-terminal microsequencing showed that 80- and 43-kDa proteins were identified as the heterodimeric nuclear factor of activated T cells (NF90/NF45) and 100 kDa as a molecular chaperone, the GRP94. The heterodimeric factor interacted preferentially with the upper-bulge region of HBV epsilon RNA helping the HBV polymerase bind the lower-bulge region. Using in vitro protein priming analysis, the initial oligonucleotide of the protein-priming product was deduced as 5'-GAAC-3', which is the complementary sequence of both regions of DR1 and epsilon in the pregenomic RNA. Previously, we also proposed that the GRP94 was associated with HBV polymerase in the human liver cell HepG2. These results suggest that the heterodimeric factor plays an important role in the priming activity of HBV polymerase.

Identification and classification of S haplotypes in Raphanus sativus by PCR-RFLP of the S locus glycoprotein (SLG) gene and the S locus receptor kinase (SRK) gene.

Polymorphism of the S-locus glycoprotein (SLG) and S-locus receptor kinase (SRK) genes in Raphanus sativus was analyzed by PCR-RFLP using SLG- and SRK-specific primers. Twenty four inbred lines of R. sativus could be grouped into nine S haplotypes. DNA fragments of SLG alleles specifically amplified from five S haplotypes by PCR with Class-I SLG-specific primers showed different profiles upon polyacrylamide-gel electrophoresis after digestion with restriction endonucleases. The five R. sativus SLG alleles were determined for their nucleotide sequences of DNA fragments. Comparison of the amino-acid sequences with a reported Brassica SLG (S(6)) showed 77-84% homology. Deduced amino-acid sequences showed 12-conserved cystein residues and three hypervariable regions which are characteristic of Brassicsa SLG. A DNA fragment was also amplified by PCR from two of each S haplotype with Class-II SLG-specific primers, and showed polymorphism when cleaved with restriction endonucleases. The nucleotide sequences of amplified DNA fragments of the Class-II SLG revealed about 60% similarity with those of the Class-I SLG. It is concluded that there exist both Class I and Class II S alleles in R. sativus, as in Brassica campestris and Brassica oleracea. PCR using SRK-specific primers amplified a DNA fragment of about 1.0 kb from seven of each S haplotype out of 24 tested. These DNA fragments showed high polymorphism in polyacrylamide-gel electrophoresis after digestion with restriction endonucleases. Nucleotide sequences of the DNA fragments amplified from the seven S haplotypes showed that the fourth and the fifth exons of SRK are highly conserved, and that there is high variation in the fifth intron, the sixth intron and seventh exon of the SRK which may be responsible for the polymorphic band patterns in PCR-RFLP analysis. The PCR-RFLP method has proven useful for the identification of S alleles in inbred lines and for listing S haplotypes in R. sativus. Phylogenic analysis of the SLG and SRK sequences from Raphanus and Brassica revealed that the Raphanus SLGs and SRKs did not form an independent cluster, but were dispersed in the tree, clustering together with Brassica SLGs and SRKs. Furthermore, SLGs and SRKs from Raphanus were both grouped into Class-I or Class-II S haplotypes. Therefore, these results suggest that the diversification of the SLG and SRK alleles occurred prior to the differentiation of the two genera Brassica and Raphanus.