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Organ culture systems are useful for elucidating the process of testicular differentiation from mammalian undifferentiated genetically male gonads, as they permit various experiments, including experiments involving the control of gene expression. However, without addition of testicular differentiation-related factors, it is difficult to induce the formation of testis cord from immature gonads by a time point earlier 12 tail somites (ts) that corresponding to 11.0 days post coitum (dpc). In this study, we attempted to establish an organ culture system that induces testis formation from immature gonads (around 8 ts: 10.5 dpc) just before Sry (sex-determining region of the Y chromosome) expression. A paired gonad-mesonephros complex of around 8 ts was placed in the groove of an agarose gel block and put the semi-cylindrical piece of agarose gel to maintain the gonad morphology. The gonads were cultured in the gas phase for 96 hr. As a result, testis cord-like structures appeared in many genetically male gonads. Cells expressing the Sertoli cell markers Sox9 (SRY-box 9) and Amh (anti-Mullerian hormone) were observed, while granulosa cell marker Foxl2 (forkhead box L2) was not detected. In addition, Sox9- and Amh-expressing cells were observed throughout the entire gonad in many individuals. Amh mRNA expression was also upregulated. Surprisingly, formation of a partial testicular structure was observed from more immature gonads (6 ts). These results show that our gonadal organ culture system is useful for elucidating the regulation mechanism of Sry expression in undifferentiated bipotential gonads.

C57BL/6J-XYPOS (B6J-XYPOS) mice, which have the Y chromosome derived from Mus musculus poschiavinus on a B6J genetic background, form ovotestes or ovaries. Previously, we replaced the genetic background of B6J-XYPOS mice with B6N and found that individuals with testes also appeared in addition to those with ovaries or ovotestes. To investigate the effect of the B6J genetic sequence on the testis differentiation, the genetic background of B6N-XYPOS mice was replaced with B6J again. The recovery of the B6J genetic background significantly decreased the incidence of testes; only ovaries developed. These results indicate that the testicular differentiation process tends to be perturbed especially in the B6J substrain. This shows the importance of substrain differences in mice usually treated as B6 collectively.

Stable reference genes are important for gene expression analyses such as quantitative PCR. The stability of 15 candidate reference genes that can be used to developing mouse gonads was thoroughly verified using combinations of multiple algorithms. The expression of these genes fluctuated greatly depending on the analysis period and/or gender. Peptidylprolyl isomerase A (Ppia) and polymerase (RNA) II (DNA directed) polypeptide A (Polr2a) were the reference genes that were used stably for a wide analysis period in developing mouse gonads. Furthermore, the stable reference genes corresponding to the analysis period and/or gender were ranked. These results are useful for the selection of the optimal reference gene required for high-precision measurements.

C57BL/6J-XYPOS (B6J-XYPOS) mice, which have the Y chromosome derived from Mus musculus poschiavinus on a B6J genetic background, form ovotestes or ovaries. Previously, we replaced the genetic background of B6J-XYPOS mice with B6N and found that individuals with testes also appeared in addition to those with ovaries or ovotestes. To investigate the effect of the B6J genetic sequence on the testis differentiation, the genetic background of B6N-XYPOS mice was replaced with B6J again. The recovery of the B6J genetic background significantly decreased the incidence of testes; only ovaries developed. These results indicate that the testicular differentiation process tends to be perturbed especially in the B6J substrain. This shows the importance of substrain differences in mice usually treated as B6 collectively.

Preeclampsia is the most common hypertensive disorder to occur during pregnancy. A healthy 38-year-old primipara presented with pretibial edema at 33 weeks of gestation followed by the development of proteinuria at 36 weeks of gestation. She had no past medical history of hypertension and was normotensive during gestation. Her proteinuria persisted after delivery, and she was also hypoalbuminemic. A renal biopsy revealed a remodeling of the glomerular basement membrane (GBM) with double contours. Some of the glomerular segments showed endothelial swelling. Immunoperoxidase staining for C4b-binding protein was positive and Protein S was weakly detected in the GBM. Electron microscopy revealed an expansion of the subendothelial zone as well as mesangial cell interposition. This case suggests that glomerular endotheliosis may therefore sometimes be present despite the absence of hypertension.

Gitelman's syndrome (GS) is an autosomal recessive disorder; it is rarely inherited over several generations. A 16-year-old boy showed hypokalemia and hypocalciuria. Clinically, he was diagnosed as GS because of diuretic responsiveness to furosemide but not thiazide. Genetic testing disclosed he was a compound heterozygote (T180K/V677M) for the SLC12A3 gene. Unexpectedly, the patient's father also showed hypokalemia and hypocalciuria. The genetic analysis showed he had an L849H mutation in addition to T180K. The present pedigree showed an extremely rare case. Diuretic tests are useful diagnostic methods, and genetic testing is necessary for precise evaluation of complicated cases as in this family.

The design and characterization of a hydrophobic cavity in de novo designed proteins provides a wide range of information about the functions of de novo proteins. We designed a de novo tetrameric coiled-coil protein with a hydrophobic pocketlike cavity. Tetrameric coiled coils with hydrophobic cavities have previously been reported. By replacing one Leu residue at the a position with Ala, hydrophobic cavities that did not flatten out due to loose peptide chains were reliably created. To perform a detailed examination of the ligand-binding characteristics of the cavities, we originally designed two other coiled-coil proteins: AM2, with eight Ala substitutions at the adjacent a and d positions at the center of a bundled structure, and AM2W, with one Trp and seven Ala substitutions at the same positions. To increase the association of the helical peptides, each helical peptide was connected with flexible linkers, which resulted in a single peptide chain. These proteins exhibited CD spectra corresponding to superhelical structures, despite weakened hydrophobic packing. AM2W exhibited binding affinity for size-complementary organic compounds. The dissociation constants, K(d), of AM2W were 220 nM for adamantane, 81 microM for 1-adamantanol, and 294 microM for 1-adamantaneacetic acid, as measured by fluorescence titration analyses. Although it was contrary to expectations, AM2 did not exhibit any binding affinity, probably due to structural defects around the designed hydrophobic cavity. Interestingly, AM2W exhibited incremental structure stability through ligand binding. Plugging of structural defects with organic ligands would be expected to facilitate protein folding.