Detection of mutations of the RB1 gene in retinoblastoma patients by using exon-by-exon PCR-SSCP analysis.

Most sporadic cases of retinoblastoma, malignant eye tumor of children, may require the identification of a mutation of the retinoblastoma gene (RB1 gene) for precise genetic counseling. We established a mutation detection system of and screened for the RB1 gene mutation in 24 patients with retinoblastoma--12 bilateral patients and 12 unilateral patients. Mutation analysis was performed by PCR-mediated SSCP analysis in the entire coding region and promoter region, as an initial screening method, followed by direct genomic sequencing. Possible oncogenic mutations were identified in 14 (58%) of 24 tumors, of which 6 were single base substitutions, 4 were small deletions, 3 were small insertions, and 1 was a complex alteration due to deletion-insertion. A constitutional somatic mosaicism was suggested in one bilateral patient. A majority (57%) of mutations were found in E1A binding domains, and all were presumed to truncate the normal gene products. The mutation analysis presented here may provide a basis for the screening system of RB1 gene mutations in retinoblastoma patients.     

Soybean Lipoxygenase L-4, a Component of the 94-kilodalton Storage Protein in Vegetative Tissues: Expression and Accumulation in Leaves Induced by Pod Removal and by Methyl Jasmonate

A lipoxygenase L-4 gene was isolated from a soybean genomiclibrary. The amino acid sequence of lipoxygenase L-4 is highlyhomologous with the partial amino acid sequence of the 94-kDavegetative storage protein, vsp94, found in paraveinal mesophyllcells in the leaves of depodded soybean plants. No L-4 expressionwas observed in maturing seeds. The L-4 gene is highly expressedin the vegetative tissues of young seedlings, including cotyledons,hypocotyls, roots and primary leaves. L-4 expression followedthe same pattern as lipoxygenase activity in cotyledons peaking3 to 5 days after germination, and returning to a basal levelby 9 days after germination. L-4 gene expression was low inthe roots, stems and leaves of 10-week-old plants. Exposureof 4-week-old plants to atmospheric methyl jasmonate increasedL-4 mRNA in leaves. Continuous pod removal from 7-week-old plantsover a 2 week period resulted in dramatic accumulation of L-4mRNA in leaves. Accumulation of the L-4 protein and three otherlipoxygenase fractions in the leaves of depodded plants wasdemonstrated by ion exchange chromatography. These results indicatethat lipoxygenase L-4 is a component of vsp94. (Received May 31, 1993; Accepted August 9, 1993)     

The kinetics of end-product inhibition of l-lactate production from xylose and glucose by Lactococcus lactis IO-1

Summary The relative contributions of lactate inhibition and the generation of sterile (undividing) cells to the low xylose utilisation rate of Lactococcus lactis IO-1 was investigated. The lactate inhibition constant of xylose grown cells was shown to be 9.3 times more than that of glucose grown cells. However, the sterile cell production rate and LDH inactivation rate of the xylose cultures were at least 10 times less than the glucose cultures. Thus, it is suggested that the slower substrate consumption rate in xylose medium is caused mainly by the large inhibition constant for the end product.     

Evolution of the therian face through complete loss of the premaxilla

The anatomical framework of the jawbones is highly conserved among most of the Osteichthyes, including the tetrapods. However, our recent study suggested that the premaxilla, the rostralmost upper jaw bone, was rearranged during the evolution of therian mammals, being replaced by the septomaxilla at least in the lateral part. In the present study, to understand more about the process of evolution from the ancestral upper jaw to the therian face, we re-examined the development of the therian premaxilla (incisive bone). By comparing mouse, bat, goat, and cattle fetuses, we confirmed that the therian premaxilla has dual developmental origins, the lateral body and the palatine process. This dual development is widely conserved among the therian mammals. Cell-lineage-tracing experiments using Dlx1-CreER^T2 mice revealed that the palatine process arises in the ventral part of the premandibular domain, where the nasopalatine nerve distributes, whereas the lateral body develops from the maxillary prominence in the domain of the maxillary nerve. Through comparative analysis using various tetrapods, we concluded that the palatine process should not be considered part of the ancestral premaxilla. It rather corresponds to the anterior region of the vomerine bone of nonmammalian tetrapods. Thus, the present findings indicate that the true premaxilla was completely lost during the evolution of the therian mammals, resulting in the establishment of the unique therian face as an evolutionary novelty. Reconsideration of the homological framework of the cranial skeleton based on the topographical relationships of the ossification center during embryonic development is warranted.     

The role of p63 in embryonic external genitalia outgrowth in mice

Embryonic external genitalia (genital tubercle [GT]) protrude from the cloaca and outgrow as cloacal development progresses. Individual gene functions and knockout phenotypes in GT development have been extensively analyzed; however, the interactions between these genes are not fully understood. In this study, we investigated the role of p63, focusing on its interaction with the Shh–Wnt/Ctnnb1–Fgf8 pathway, a signaling network that is known to play a role in GT outgrowth. p63 was expressed in the epithelial tissues of the GT at E11.5, and the distal tip of the GT predominantly expressed the ΔNp63α isoform. The GTs in p63 knockout embryos had normal Shh expression, but CTNNB1 protein and Fgf8 gene expression in the distal urethral epithelium was decreased or lost. Constitutive expression of CTNNB1 in p63-null embryos restored Fgf8 expression, accompanied by small bud structure development; however, such bud structures could not be maintained by E13.5, at which point mutant GTs exhibited severe abnormalities showing a split shape with a hemorrhagic cloaca. Therefore, p63 is a key component of the signaling pathway that triggers Fgf8 expression in the distal urethral epithelium and contributes to GT outgrowth by ensuring the structural integrity of the cloacal epithelia. Altogether, we propose that p63 plays an essential role in the signaling network for the development of external genitalia.     

Increased anti-oxidative action compensates for collagen tissue degeneration in vitiligo dermis

Vitiligo is a common depigmentation disorder characterized by the selective loss of melanocytes. In our daily clinic experience, we noticed that the skin tightness of hypopigmented lesions would be more evident in comparison to that of uninvolved perilesional skin in vitiligo patients. Therefore, we hypothesized that collagen homeostasis might be maintained in vitiligo lesions, irrespective of the substantial excessive oxidative stress that occurs in association with the disease. We found that the expression levels of collagen-related genes and anti-oxidative enzymes were upregulated in vitiligo-derived fibroblasts. Abundant collagenous fibers were observed in the papillary dermis of vitiligo lesions in comparison to uninvolved perilesional skin by electron microscopy. The production of matrix metalloproteinases that degraded collagen fibers was suppressed. The deposition of acrolein adduct protein, which is a product of oxidative stress, was significantly reduced in vitiligo dermis and fibroblasts. As part of the mechanism, we found upregulation of the NRF2 signaling pathway activity, which is an important defense system against oxidative stress. Taken together, we demonstrated that the anti-oxidative action and collagen production were upregulated and that the collagen degeneration was attenuated in vitiligo dermis. These new findings may provide important clues for the maintenance of antioxidant ability in vitiligo lesions.     

Effect of gravity on apical dominance in Pharbitis nil.

When the upper part of main shoot of morning glory (Pharbitis nil) is gently bent down, lateral bud on the bending region is released from apical dominance and starts to elongate. But, clinorotating the bending shoots prevents the release of the lateral bud from apical dominance. These results suggest that gravity affects apical dominance in morning glory. Here we verified the gravity-regulated apical dominance by using a weeping morning glory defective in gravitropic response due to abnormal differentiation of endodermis. That is, bending main shoot of the weeping morning glory hardly caused the lateral bud to elongate. In addition, decapitation of apical bud released the lateral bud from apical dominance, and exogenous auxin applied to the cut surface of the decapitated stem was inhibitory to the outgrowth of the lateral bud in the wild type. However, the effect of auxin was much less in the weeping morning glory. Thus, apical dominance of the weeping morning glory was weaker and less influenced by gravity than that of the wild type, which could occur due to abnormal differentiation of endodermis required for graviperception.     

Effect of gravity stress on fidelity of DNA double-strand break repair.

DNA double strand break (DSB) causes many cytotoxic effects such as cellular lethality, somatic mutation, and carcinogenesis. Fidelity of DSB repair is a important factor that determines the quality of genomic stability. It is known that the most of DSBs are properly repaired on the earth, however, little is known whether those are rejoined at the same fidelity even under the space environment. One of the DSB repair pathway, homologous recombination (HR), allows the cells to repair their DSBs with error free. Therefore, the efficiency of HR is a good index to assess the fidelity of DSB repair. In order to clarify the effect of gravity stress on HR pathway, we established a cell line that can detect a site-specific DNA repair via HR. The cells carrying a reporter construct for HR were incubated under hypergravity condition after induction of site specific DSB. Our preliminary results suggest that the gravity stress may affect the HR efficiency.