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SOX9 haploinsufficiency underlies campomelic dysplasia (CD) with or without testicular dysgenesis. Current understanding of the phenotypic variability and mutation spectrum of SOX9 abnormalities remains fragmentary. Here, we report three patients with hitherto unreported SOX9 abnormalities. These patients were identified through molecular analysis of 33 patients with 46,XY disorders of sex development (DSD). Patients 1–3 manifested testicular dysgenesis or regression without CD. Patients 1 and 2 carried probable damaging mutations p.Arg394Gly and p.Arg437Cys, respectively, in the SOX9 C‐terminal domain but not in other known 46,XY DSD causative genes. These substitutions were absent from ~120,000 alleles in the exome database. These mutations retained normal transactivating activity for the Col2a1 enhancer, but showed impaired activity for the Amh promoter. Patient 3 harbored a maternally inherited ~491 kb SOX9 upstream deletion that encompassed the known 32.5 kb XY sex reversal region. Breakpoints of the deletion resided within nonrepeat sequences and were accompanied by a short‐nucleotide insertion. The results imply that testicular dysgenesis and regression without skeletal dysplasia may be rare manifestations of SOX9 abnormalities. Furthermore, our data broaden pathogenic SOX9 abnormalities to include C‐terminal missense substitutions which lead to target‐gene‐specific protein dysfunction, and enhancer‐containing upstream microdeletions mediated by nonhomologous end‐joining.

Our previous studies revealed that the double variant of cytochrome P450 (CYP)105A1, R73V/R84A, has a high ability to convert vitamin D(3) to its biologically active form, 1α,25-dihydroxyvitamin D(3) [1α,25(OH)(2)D(3)], suggesting the possibility for R73V/R84A to produce 1α,25(OH)(2)D(3). Because Actinomycetes, including Streptomyces, exhibit properties that have potential advantages in the synthesis of secondary metabolites of industrial and medical importance, we examined the expression of R73V/R84A in Streptomyces lividans TK23 cells under the control of the tipA promoter. As expected, the metabolites 25-hydroxyvitamin D(3) [25(OH)D(3)] and 1α,25(OH)(2)D(3) were detected in the cell culture of the recombinant S. lividans. A large amount of 1α,25(OH)(2)D(3), the second-step metabolite of vitamin D(3), was observed, although a considerable amount of vitamin D(3) still remained in the culture. In addition, novel polar metabolites 1α,25(R),26(OH)(3)D(3) and 1α,25(S),26(OH)(3)D(3), both of which are known to have high antiproliferative activity and low calcemic activity, were observed at a ratio of 5:1. The crystal structure of the double variant with 1α,25(OH)(2)D(3) and a docking model of 1α,25(OH)(2)D(3) in its active site strongly suggest a hydrogen-bond network including the 1α-hydroxyl group, and several water molecules play an important role in the substrate-binding for 26-hydroxylation. In conclusion, we have demonstrated that R73V/R84A can catalyze hydroxylations at C25, C1 and C26 (C27) positions of vitamin D(3) to produce biologically useful compounds.

Summary The plant genome contains a large number of disease resistance (R) genes that have evolved through diverse mechanisms. Here, we report that a long terminal repeat (LTR) retrotransposon contributed to the evolution of the rice blast resistance gene Pit. Pit confers race-specific resistance against the fungal pathogen Magnaporthe grisea, and is a member of the nucleotide-binding site leucine-rich repeat (NBS-LRR) family of R genes. Compared with the non-functional allele PitNpb, the functional allele PitK59 contains four amino acid substitutions, and has the LTR retrotransposon Renovator inserted upstream. Pathogenesis assays using chimeric constructs carrying the various regions of PitK59 and PitNpb suggest that amino acid substitutions might have a potential effect in Pit resistance; more importantly, the upregulated promoter activity conferred by the Renovator sequence is essential for Pit function. Our data suggest that transposon-mediated transcriptional activation may play an important role in the refunctionalization of additional ‘sleeping’R genes in the plant genome.

This study was conducted to examine the regeneration process of hepatic nerves following surgical hepatic denervation in dogs. A denervation model was surgically created by removing all visible nerves around the hepatoduodenal ligament along with the peri-hepatic tissues. The hepatic nerves were examined on the hepatic specimens taken at 1 week, 1 month and 3 months post-denervation by means of immunohistochemical staining, and also electron microscopy. At 1 week post-denervation, the extrinsic hepatic nerves were observed not to have regenerated. However, at 1 month post-denervation, GAP-43-positive nerves were identified and regenerating axons were shown to be present on electron microscopic observation. The immunoreactivity for anti-GAP-43 antibody was not shown any longer at 3 months post-denervation, and the regenerated nerve axons were shown to be similar to those at pre-denervation on ultrastructural study. Results of the present study suggested that regeneration of the extrinsic hepatic nerves began to appear about 1 month after the hepatic denervation, and was completed by 3 months post-denervation.

The effects of bisphenol A (BPA) on UDP-glucuronosyltransferase 1A6 (UGT1A6) activities in microsomes from human livers and yeast cells expressing human UGT1A6 (humUGT1A6) were investigated. Serotonin (5-HT) and 4-methylumbelliferone (4-MU) were used as the substrates for UGT1A6. BPA dose-dependently inhibited 5-HT and 4-MU glucuronidation activities in both enzyme sources. The IC(50) values of BPA for 5-HT and 4-MU glucuronidation activities were 156 and 163 microM for liver microsomes, and 84.6 and 80.3 microM for yeast cell microsomes expressing humUGT1A6, respectively. The inhibitory pattern of BPA for 5-HT and 4-MU glucuronidation activities in human liver microsomes exhibited a mixture of competitive and noncompetitive components, with K(i) values of 84.9 and 72.3 microM, respectively. In yeast cell microsomes expressing humUGT1A6, 5-HT glucuronidation activities were noncompetitively inhibited by BPA (K(i) value, 65.5 microM), whereas the inhibition of 4-MU glucuronidation activities by BPA exhibited the mixed type (K(i) value, 42.5 microM). These results suggest that BPA interacts with human UGT1A6 enzyme, and that the interaction may contribute to the toxicity, such as hormone disruption and reproductive effects, of BPA.

Summary. T-cell granular lymphocyte-proliferative disorder (T-GLPD) is characterized by the proliferation of cytotoxic T lymphocytes, and is often associated with pure red cell aplasia (PRCA). The mechanism involved in the development of PRCA in T-GLPD is unknown. Peripheral blood mononuclear cells were isolated from 20 patients with T-GLPD. Ten patients had associated PRCA. Granular lymphocytes (GLs) of T-GLPD are positive for CD94, but not NKG2A. To clarify the functional role of CD94 in T-GLPD, we performed a cytotoxicity assay against the trophoblast cell line, BeWo, which is known to express human leucocyte antigen (HLA)-E, a natural ligand of CD94, and is deficient in other HLA class I and class II antigens. GLs isolated from T-GLPD with PRCA patients killed BeWo cells more significantly than GLs from T-GLPD without PRCA patients. Furthermore, GLs from T-GLPD with PRCA were significantly stimulated by a monoclonal antibody against CD94, whereas those of T-GLPD without PRCA were not. Taken together, HLA-E, a ligand of CD94, was suggested to stimulate CD94+ cells to kill HLA-E+ cells in T-GLPD with PRCA. GLs of T-GLPD with PRCA have a potential positive activity against HLA-E+ cells, whereas GLs from T-GLPD without PRCA do not. CD94 may play a key role in the pathogenesis of PRCA in T-GLPD.

We treated rapidly growing Jurkat cells with 40 nmol/l of doxorubicin for 72 h. After 36 h, the G2-arrested cells became larger and some of them started endoreplication. Nuclear staining with Hoechst 33342 combined with propidium iodide (PI) exclusion revealed that about 90% of the cells were necrotic at 72 h, although apoptotic cells accounted for only 8%. Incubation with 40 nmol/l of aclarubicin or cytosine beta-d-arabinofuranoside for 60 h induced necrosis both in Jurkat and ml-1 cells. Pre-necrotic Jurkat cells incubated with 40 nmol/l of doxorubicin had much higher intracellular reactive oxygen species (ROS) levels than pre-apoptotic ones. Addition of Tempol or Desferal accelerated doxorubicin-induced necrosis and partially converted it into apoptosis. Both antioxidants reduced surviving colony numbers of prenecrotic Jurkat cells. n-acetyl-l-cysteine had little effect on the apoptotic conversion but profoundly accelerated necrosis. Because an apoptosis-resistant Jurkat subclone was also refractory to doxorubicin-induced necrosis, apoptosis and necrosis might share some common pathways. Low-dose doxorubicin increased micronuclei-positive cell percentages and also suppressed high-dose doxorubicin-induced apoptosis in Jurkat and ml-1 cells. Some of the prenecrotic cells, therefore, might survive and obtain genomic instability. Antioxidants may be useful to suppress, at least to some extent, this vicious consequence.

Background/aims Because little has been known about the morphological and functional consequences of liver transplantation on hepatic autonomic nerves, we examined the time-course of extrinsic hepatic innervation at the level of the porta hepatis of liver allografts. Methods Orthotopic liver transplantation was performed using male Lewis rats. Crosscut tissue specimens were obtained postoperatively for up to 6 months from the porta hepatis of transplanted livers, and processed for immunohistochemical staining for protein gene product 9.5 (PGP 9.5) and growth-associated protein 43 (GAP-43), and for transmission electron microscopy (TEM). Results Extrinsic nerve fibers at the porta hepatis stained positively for PGP 9.5 throughout the entire study period. In contrast, the immunoreactivity of GAP-43 was negative at postoperative day (POD) 1 and 2. GAP-43-positive nerves were first observed to appear in the porta hepatis at POD 3. The immunoreactivity of GAP-43 remained positive thereafter until 3 months post-OLT, and became negative in all the specimens at 4 months post-OLT. Transmission electron microscopy demonstrated a small number of regenerating axons existing among many degenerating axons at POD 3. At 3 months post-OLT, most regenerating axons had been fully ensheathed by the cytoplasm of Schwann cells, although their density remained at a lower level compared with normal. Conclusion The results of this study suggest that liver allografts become extrinsically reinnervated, with the regenerating axons reaching the hepatic hilus 3 days after transplantation. The process of extrinsic hepatic reinnervation is considered to almost terminate 4 months after transplantation in rats.