A comparison study on ribonuclease A modifications induced by substituted p-benzoquinones.

In this paper, we present our investigation on ribonuclease A (RNase) modifications induced by 1,4-benzoquinone (PBQ), 2-methyl-1,4-benzoquinone (MBQ), and 2-chloro-1,4-benzoquinone (CBQ). The goal of the study was to evaluate quinone-induced protein modifications as well as substituent effects, utilizing several techniques such as SDS-PAGE, fluorescence spectroscopy, microscopy, and LC-ESI(+)-QTOF-MS. SDS-PAGE experiments revealed that all quinones modify RNase through oligomerization as well as polymeric aggregation; with CBQ functioning as the most efficient quinone while MBQ was least efficient. The fluorescence emission was found to be less intense and the anisotropy values were found to be slightly higher for the modified RNase compared to the unmodified RNase. UV-Vis spectroscopy indicated that all three quinones formed adducts in which they were covalently linked to RNase. Confocal imaging analysis showed that the presence of CBQ resulted in massive RNase aggregation, while PBQ-treated RNase formed much smaller aggregates. MBQ-treated RNase exhibited micrographic features that closely resembled those of the unmodified RNase. LC-ESI(+)-QTOF-MS studies indicated the nature of PBQ- and CBQ-induced RNase modifications are complex mainly due to simultaneously occurrence of both adduct formation and oligomerization. Kinetic studies on quinone reactivity toward lysine revealed the rank order of CBQ>PBQ≫MBQ, based on the second-order rate constants. We also utilized scanning electron microscopy in order to investigate the effect of modified RNase on the biomineralization of salts.

A reciprocal translocation dissects roles of Pax6 alternative promoters and upstream regulatory elements in the development of pancreas, brain, and eye.

Pax6 encodes a transcription factor with key roles in the development of the pancreas, central nervous system, and eye. Gene expression is orchestrated by several alternative promoters and enhancer elements that are distributed over several hundred kilobases. Here, we describe a reciprocal translocation, called 1Gso, which disrupts the integrity of transcripts arising from the 5'-most promoter, P0, and separates downstream promoters from enhancers active in pancreas and eye. Despite this fact, 1Gso animals exhibit none of the dominant Pax6 phenotypes, and the translocation complements recessive brain and craniofacial phenotypes. However, 1Gso fails to complement Pax6 recessive effects in lacrimal gland, conjunctiva, lens, and pancreas. The 1Gso animals also express a corneal phenotype that is related to but distinct from that expressed by Pax6 null mutants, and an abnormal density and organization of retinal ganglion cell axons; these phenotypes may be related to a modest upregulation of Pax6 expression from downstream promoters that we observed during development. Our investigation maps the activities of Pax6 alternative promoters including a novel one in developing tissues, confirms the phenotypic consequences of upstream enhancer disruption, and limits the likely effects of the P0 transcript null mutation to recessive abnormalities in the pancreas and specific structures of the eye.

Modifications of ribonuclease A induced by p-benzoquinone.

The nature of ribonuclease A (RNase) modifications induced by p-benzoquinone (pBQ) was investigated using several analysis methods. SDS-PAGE experiments revealed that pBQ was efficient in producing oligomers and polymeric aggregates when RNase was incubated with pBQ. The fluorescence behavior and anisotropy changes of the modified RNase were monitored for a series of incubation reactions where RNase (0.050 mM) was incubated with pBQ (0.050, 0.25, 0.50, 1.50 mM) at 37 °C in phosphate buffer (pH 7.0, 50 mM). The modified RNase exhibited less intense fluorescence and slightly higher anisotropy than the unmodified RNase. UV-Vis spectroscopy indicated that pBQ formed covalent bonds to the modified RNase. Confocal imaging analysis confirmed the formation of the polymeric RNase aggregates with different sizes upon exposure of RNase to high concentrations of pBQ. The interaction between the modified RNase and salts affecting biomineralization of salts was also investigated by scanning electron microscopy. Overall, our results show that pBQ can induce formation of both RNase adducts and aggregates thus providing a better understanding of its biological activity.

Use of flatbed transparency scanners in zebrafish research: versatile and economical adjuncts to traditional imaging tools for the Danio rerio laboratory.

Flatbed transparency scanners are typically relegated to routine office tasks, yet they do offer a variety of potentially useful imaging tools for the zebrafish laboratory. These include motility screens, oocyte maturation and egg activation assays as well as counting and measuring tasks. When coupled with Macroscheduler (http://www.mjtnet.com) and ImageJ (http://rsbweb.nih.gov/ij), the scanner becomes a stable platform for imaging large arrays of zebrafish oocytes, embryos, larvae, and adults. Such large arrays are a prerequisite to the development of high-throughput screens for small molecules as potential therapeutic drugs in the treatment of many diseases including cancer and epilepsy. Thus the scanner may have a role in adapting zebrafish to future drug and mutagenesis screening. In this chapter, some of the uses of scanners are outlined to bring attention to the potentials of this simple-to-use, flexible, inexpensive device for the zebrafish research community.

Generation of a Snail1 (Snai1) conditional null allele.

Members of the Snail gene superfamily, which encode zinc finger transcriptional repressors, play critical roles in the establishment of the vertebrate body plan. The Snail1 (Snai1) gene promotes epithelial-mesenchymal transitions during development and disease progression, and Snai1 null mouse embryos exhibit defects in gastrulation. However, the early embryonic lethality of Snai1 null embryos precludes the study of Snai1 function in other developmental contexts or diseases. To overcome this restriction, we generated a Snai1 conditional null allele by flanking the promoter and first two exons of the Snai1 gene with loxP sites. Cre-mediated deletion of the Snai1(flox) allele generates the Snai1(del2) allele, which behaves genetically as a Snai1 null allele. This conditional null allele will enable investigation of Snai1 function in a variety of developmental and pathological contexts.

Developmental transcription factor slug is required for effective re-epithelialization by adult keratinocytes.

During re-epithelialization of cutaneous wounds, keratinocytes recapitulate several aspects of the embryonic process of epithelial-mesenchymal transition (EMT), including migratory activity and reduced intercellular adhesion. The transcription factor Slug modulates EMT in the embryo and controls desmosome number in adult epithelial cells, therefore, we investigated Slug expression and function during cutaneous wound re-epithelialization. Slug expression was elevated in keratinocytes bordering cutaneous wounds in mice in vivo, in keratinocytes migrating from mouse skin explants ex vivo, and in human keratinocytes at wound margins in vitro. Expression of the related transcription factor Snail was not significantly modulated in keratinocytes during re-epithelialization in vitro. Epithelial cell outgrowth from skin explants of Slug knockout mice was severely compromised, indicating a critical role for Slug in epithelial keratinocyte migration. Overexpression of Slug in cultured human keratinocytes caused increased cell spreading and desmosomal disruption, both of which were most pronounced at wound margins. Furthermore, in vitro wound healing was markedly accelerated in keratinocytes that ectopically expressed Slug. Taken together, these findings suggest that Slug plays an important role during wound re-epithelialization in adult skin and indicate that Slug controls some aspects of epithleial cell behavior in adult tissues as well as during embryonic development.

Slug expression during organogenesis in mice.

The vertebrate Slug gene encodes a zinc finger-containing transcriptional repressor. Here we report expression of the mouse Slug gene during organogenesis and late fetal development using histochemical detection of beta-galactosidase expressed from a targeted Slug(lacZ) knock-in allele. The Slug gene is highly expressed in the mesenchymal or stromal component of numerous organs. It is also highly expressed in craniofacial mesenchyme, in bone of both mesodermal and neural crest origin, and in the outflow tract and the endocardial cushions of the heart.

Craniosynostosis in Twist heterozygous mice: a model for Saethre-Chotzen syndrome.

Saethre-Chotzen syndrome is a common autosomal dominant form of craniosynostosis, the premature fusion of the sutures of the calvarial bones of the skull. Most Saethre-Chotzen syndrome cases are caused by haploinsufficiency for the TWIST gene. Mice heterozygous for a null mutation of the Twist gene replicate certain features of Saethre-Chotzen syndrome, but have not been reported to exhibit craniosynostosis. We demonstrate that Twist heterozygous mice exhibit fusions of the coronal suture and other cranial suture abnormalities, indicating that Twist heterozygous mice constitute a better animal model for Saethre-Chotzen syndrome than was previously appreciated.

Slug is a novel downstream target of MyoD. Temporal profiling in muscle regeneration.

Temporal expression profiling was utilized to define transcriptional regulatory pathways in vivo in a mouse muscle regeneration model. Potential downstream targets of MyoD were identified by temporal expression, promoter data base mining, and gel shift assays; Slug and calpain 6 were identified as novel MyoD targets. Slug, a member of the snail/slug family of zinc finger transcriptional repressors critical for mesoderm/ectoderm development, was further shown to be a downstream target by using promoter/reporter constructs and demonstration of defective muscle regeneration in Slug null mice.