Crystallization and preliminary X-ray diffraction studies of a replication initiator protein (RepE54) of the mini-F plasmid complexed with iteron DNA.

A replication initiator protein (RepE54) complexed with iteron DNA at its binding site was crystallized by the hanging drop vapor diffusion method. The crystals belong to monoclinic space group C2 with unit cell dimensions of a = 108.4 A, b = 81.9 A, c = 73.9 A, and beta = 121.5 degrees, where one molecule of the protein-DNA complex exists per asymmetric unit. They diffract X-rays up to 2.6 A resolution with synchrotron radiation.

Kinematic magnetic resonance imaging (MRI) of the normal shoulder: assessment of the shapes and signals of the superior and inferior labra with abductive movement using an open-type imager.

A preliminary study was conducted to evaluate the superior and inferior glenoid labra with abductive movement using an open-type MR unit in asymptomatic healthy volunteers. Both fast low angle shot (FLASH) and turbo spin echo (TSE) images were obtained to evaluate the shapes of both the superior and inferior labra, as well as to assess changes in signal at these sites. As the abduction angle was increased, the shape of the superior labrum changed from round or triangular to crescentic and a higher signal was frequently seen. At an abduction angle of 150 degrees, an increase in signal was seen in one-half of the superior labra; this increase was noted more frequently in volunteers over 40 years of age. In some of the superior labra, the increase in signal seen at 150 degrees abduction disappeared on subsequent images obtained at 0 degrees abduction. Hence, the increase in signal was considered to be a reversible change. The shape of the inferior labrum tended to change from crescentic to triangular or round. An increase in signal in the inferior labrum was unrelated to the abduction angle. Abductive kinematic studies using an open-type MR unit provides information about the morphology of the superior and inferior labra, as well as information about signal changes occurring at these sites.

The human protein kinase HIPK2 phosphorylates and downregulates the methyl-binding transcription factor ZBTB4.

HIPK2 is a eukaryotic Serine-Threonine kinase that controls cellular proliferation and survival in response to exogenous signals. Here, we show that the human transcription factor ZBTB4 is a new target of HIPK2. The two proteins interact in vitro, colocalize and associate in vivo, and HIPK2 phosphorylates several conserved residues of ZBTB4. Overexpressing HIPK2 causes the degradation of ZBTB4, whereas overexpressing a kinase-deficient mutant of HIPK2 has no effect. The chemical activation of HIPK2 also decreases the amount of ZBTB4 in cells. Conversely, the inhibition of HIPK2 by drugs or by RNA interference causes a large increase in ZBTB4 levels. This negative regulation of ZBTB4 by HIPK2 occurs under normal conditions of cell growth. In addition, the degradation is increased by DNA damage. These findings have two consequences. First, we have recently shown that ZBTB4 inhibits the transcription of p21. Therefore, the activation of p21 by HIPK2 is two-pronged: stimulation of the activator p53, and simultaneous repression of the inhibitor ZBTB4. Second, ZBTB4 is also known to bind methylated DNA and repress methylated sequences. Consequently, our findings raise the possibility that HIPK2 might influence the epigenetic regulation of gene expression at loci that remain to be identified.

Requirement of FoxD3-class signaling for neural crest determination in Xenopus.

Fox factors (winged-helix transcription factors) play important roles in early embryonic patterning. We show here that FoxD3 (Forkhead 6) regulates neural crest determination in Xenopus embryos. Expression of FoxD3 in the presumptive neural crest region starts at the late gastrula stage in a manner similar to that of Slug, and overlaps with that of Zic-r1. When overexpressed in the embryo and in ectodermal explants, FoxD3 induces expression of neural crest markers. Attenuation of FoxD3-related signaling by a dominant-negative FoxD3 construct (FoxD3delN) inhibits neural crest differentiation in vivo without suppressing the CNS marker Sox2. Interestingly, these loss-of-function phenotypes are reversed by coinjecting SLUG: In animal cap explants, neural crest differentiation induced by Slug and Wnt3a is also inhibited by FoxD3delN but not by a dominant-negative form of XBF2. Loss-of-function studies using dominant-negative forms of FoxD3 and Slug indicate that Slug induction by Zic factors requires FoxD3-related signaling, and that FoxD3 and Slug have different requirements in inducing downstream neural crest markers. These data demonstrate that FoxD3 (or its closely related factor) is an essential upstream regulator of neural crest determination.

P elements and P-M characteristics in natural populations of Drosophila melanogaster in the southernmost islands of Japan and in Taiwan.

In order to study P element dynamics in natural populations of Drosophila melanogaster, 126 isofemale lines were examined from seven of the southernmost islands of Japan (the Sakishima Islands) and from Taiwan. Gonadal dysgenesis (GD) tests showed large divergences in the P-M phenotypes (P inducing and P repressing abilities) between the island populations. The P-M characteristics of each population, however, had not greatly changed in the past 15 years. Their genomic P element profiles are highly similar, consisting mostly of full-size P and of KP elements. We found no clear relationship between phenotype and genomic P element composition.

Requirement of Sox2-mediated signaling for differentiation of early Xenopus neuroectoderm.

From early stages of development, Sox2-class transcription factors (Sox1, Sox2 and Sox3) are expressed in neural tissues and sensory epithelia. In this report, we show that Sox2 function is required for neural differentiation of early Xenopus ectoderm. Microinjection of dominant-negative forms of Sox2 (dnSox2) mRNA inhibits neural differentiation of animal caps caused by attenuation of BMP signals. Expression of dnSox2 in developing embryos suppresses expression of N-CAM and regional neural markers. We have analyzed temporal requirement of Sox2-mediated signaling by using an inducible dnSox2 construct fused to the ligand-binding domain of the glucocorticoid receptor. Attenuation of Sox2 function both from the late blastula stage and from the late gastrula stage onwards causes an inhibition of neural differentiation in animal caps and in whole embryos. Additionally, dnSox2-injected cells that fail to differentiate into neural tissues are not able to adopt epidermal cell fate. These data suggest that Sox2-class genes are essential for early neuroectoderm cells to consolidate their neural identity during secondary steps of neural differentiation.

HSF3 is a major heat shock responsive factor duringchicken embryonic development.

The expression of heat shock genes in vertebrates is regulated mainly at the level of transcription by four heat shock transcription factors (HSFs 1-4). Avian cells express at least three HSFs (HSFs 1-3). HSF1 is rapidly activated by even mild heat shock, while HSF3 is activated only by severe heat shock. In contrast, HSF2 is not activated by heat stress and has been speculated to have developmental functions. Here, we examined the temporal and spatial profiles of changes in the levels of these three HSFs in various tissues during chicken development. We found that HSF3 was almost constantly expressed in various tissues during early to late chicken development. The expression of HSF1 was equally high in most tissues early in development and thereafter declined to different levels in a tissue-dependent manner. Thus, HSF3 became the dominant heat-responsive factor in all tissues examined. The magnitude of heat shock response determined by Northern blotting did not always correlate with the level of HSF1 expression, suggesting that not only HSF1 but also HSF3 may be a major factor mediating stress signals to heat shock gene expression in the chicken. In addition, the high-level and ubiquitous expression of HSF2 as well as HSF1 and HSF3 in early embryogenesis suggested the involvement of these factors in all developmental processes.

The mammalian HSF4 gene generates both an activator and a repressor of heat shock genes by alternative splicing.

The expression of heat shock genes is controlled at the level of transcription by members of the heat shock transcription factor family in vertebrates. HSF4 is a mammalian factor characterized by its lack of a suppression domain that modulates formation of DNA-binding homotrimer. Here, we have determined the exon structure of the human HSF4 gene and identified a major new isoform, HSF4b, derived by alternative RNA splicing events, in addition to a previously reported HSF4a isoform. In mouse tissues HSF4b mRNA was more abundant than HSF4a as examined by reverse transcription-polymerase chain reaction, and its protein was detected in the brain and lung. Although both mouse HSF4a and HSF4b form trimers in the absence of stress, these two isoforms exhibit different transcriptional activity; HSF4a acts as an inhibitor of the constitutive expression of heat shock genes, and hHSF4b acts as a transcriptional activator. Furthermore HSF4b but not HSF4a complements the viability defect of yeast cells lacking HSF. Moreover, heat shock and other stresses stimulate transcription of target genes by HSF4b in both yeast and mammalian cells. These results suggest that differential splicing of HSF4 mRNA gives rise to both an inhibitor and activator of tissue-specific heat shock gene expression.

Bile duct stenosis due to portal cavernomas: MR portography and MR cholangiopancreatography demonstration.

We report two cases of bile duct stenosis due to portal cavernomas. Smooth stenoses were seen arising from both walls of the common bile duct on magnetic resonance (MR) cholangiopancreatography. On contrast-enhanced MR portography, peribiliary tortuous vessels were evident, indicating portal cavernomas. MR imaging can evaluate the biliary tree and portal systems noninvasively and was useful for evaluating this condition.

[Evaluation of the cystic duct and cysticohepatic junction with MR cholangiography: comparison of various techniques and clinical evaluation with respiratory-triggered three-dimensional multislab technique].

The purpose of this study was to evaluate depiction of the cystic duct and cysticohepatic junction by MR cholangiography (MRC). In 10 volunteers, MR cholangiograms were obtained by breath-hold two-dimensional (2D), respiratory-triggered 2D, respiratory-triggered three-dimensional (3D) single slab, and 3D multislab techniques. The images then were compared qualitatively. MRC using the respiratory-triggered 3D multislab techniques was evaluated as better than the other techniques, and was performed in 35 patients. Depiction of the anatomy of the cystic duct and cysticohepatic junction were evaluated. In 8 of 35 patients, MRC images were compared with those obtained by endoscopic retrograde cholangiography (ERC). The cystic duct and cysticohepatic junction were visualized adequately in 93% of volunteers and patients by the respiratory-triggered 3D multislab technique. Anatomic variations in the cystic duct and cysticohepatic junction were evaluated. The frequency of anatomic variations was the same as previously reported. The anatomic evaluations obtained by MRC were correlated closely with those obtained by ERC in 8 patients. In conclusion, MRC with the respiratory-triggered 3D multislab techniques is useful in evaluation of the cystic duct and cysticohepatic junction.