Head position affects the direction of occlusal force during tapping movement.

Despite numerous reports describing the relationship between head position and mandibular movement in human subjects, the direction and magnitude of force at the occlusal contacts have not been investigated in relation to head position. The objective was to investigate the effect of head position on the direction of occlusal force while subjects performed a tapping movement. Twenty-three healthy adult subjects were asked to sit on a chair with their back upright and to perform 15 tapping movements in five different head positions: natural head position (control); forward; backward; and right and left rolled. The direction and magnitude of force were measured using a small triaxial force sensor. The Wilcoxon signed-rank test and Bonferroni test were used to compare head positions in each angle of the anteroposterior axis direction and the lateral axis direction with respect to the superior axis. The force element in the anteroposterior axis shifted to the forward direction in the head position pitched backward, compared with control, pitched forward and rolled left positions (P = .02, <.01 and <.01, respectively). The force direction in the lateral axis with the head position rolled to the right or left shifted to the left and right directions, respectively, compared with those in the other positions (P < .05). Results of this study suggest that the head should be maintained in a position in which a stable tapping movement can be performed in a relaxed position without anteroposterior and lateral loading.

The long terminal repeat negative control region is a critical element for insertional oncogenesis after gene transfer into hematopoietic progenitors with Moloney murine leukemia viral vectors.

Integrating vectors based on γ-retroviruses and containing full-length long terminal repeats (LTRs) have been associated with activation of oncogene expression and leukemogenesis in human gene therapy trials. Identification of the specific molecular elements of the LTRs that have a role in insertional oncogenesis events is important as it can lead to the development of safer gene transfer vectors. The negative control region (NCR) of the LTR is a particularly well-conserved sequence among mammalian γ-retroviruses with demonstrated regulatory activity of gene transcription in hematopoietic cells, which led us to hypothesize that this region may have a role in insertional oncogenesis after γ-retroviral vector (GV)-mediated gene transfer into hematopoietic progenitors. We used an in vitro assay of murine bone marrow cell immortalization to compare the immortalization capabilities of a series of GVs carrying murine leukemia virus (MLV) LTR deletion mutants. Compared with GV carrying the full-length MLV LTR, deletion of the complete LTR enhancer sequence showed significant reduction of immortalization rates. However, the use of a mutant LTR deleted of the enhancer sequence, with exception of the NCR, did not affect immortalization. Importantly, the inclusion of an LTR mutant devoid only of the NCR did show significant reduction of immortalization rates compared with the full LTR sequence. Therefore, our data point to the NCR as a key element for immortalization and justify additional studies to evaluate its specific role in MLV-mediated insertional oncogenesis.

p63(TP63) elicits strong trans-activation of the MFG-E8/lactadherin/BA46 gene through interactions between the TA and DeltaN isoforms.

We report here that human MFGE8 encoding milk fat globule-EGF factor 8 protein (MFG-E8), also termed 46 kDa breast epithelial antigen and lactadherin, is transcriptionally activated by p63, or TP63, a p53 (TP53) family protein frequently overexpressed in head-and-neck squamous cell carcinomas, mammary carcinomas and so on. Despite that human MFG-E8 was originally identified as a breast cancer marker, and has recently been reported to provide peptides for cancer immunotherapy, its transcriptional control remains an open question. Observations in immunohistochemical analyses, a tetracycline-induced p63 expression system and keratinocyte cultures suggested a physiological link between p63 and MFGE8. By reporter assays with immediately upstream regions of MFGE8, we determined that the trans-activator (TA) isoforms of p63 activate MFGE8 transcription though a p53/p63 motif at -370, which was confirmed by a chromatin immunoprecipitation experiment. Upon siRNA-mediated p63 silencing in a squamous cell carcinoma line, MFG-E8 production decreased to diminish Saos-2 cell adhesion. Interestingly, the DeltaN-p63 isoform lacking the TA domain enhanced the MFGE8-activating function of TA-p63, if DeltaN-p63 was dominant over TA-p63 as typically observed in undifferentiated keratinocytes and squamous cell carcinomas, implying a self-regulatory mechanism of p63 by the TA:DeltaN association. MFG-E8 may provide a novel pathway of epithelial-nonepithelial cell interactions inducible by p63, probably in pathological processes.

Characterization of binding and utilization of hemoglobin by Prevotella nigrescens.

The ability of Prevotella nigrescens to utilize and bind to hemoglobin was investigated. Growth studies showed that P. nigrescens was able to utilize hemoglobin efficiently as an iron source. Binding of P. nigrescens to hemoglobin was demonstrated by dot blot assay. Heat and trypsin treatments of the bacteria led to a decrease in activity. Globin gave nearly complete inhibition of activity. Additionally, lactoferrin partially inhibited activity. In contrast, transferrin, cytochrome C and catalase exerted little or no inhibitory effect. Although the sugars tested did not affect activity, several of the amino acids tested, including arginine, cysteine, histidine and lysine, inhibited activity. In a solid phase assay, 41-, 56- and 59-kDa proteins of P. nigrescens reacted with hemoglobin. These results suggest that P. nigrescens utilizes hemoglobin for growth and 41-, 56- and 59-kDa proteins may be involved in hemoglobin binding.

Design and development of a catalytic ribonucleoprotein.

Ribonucleoproteins (RNPs) consisting of derivatives of a ribozyme and an RNA-binding protein were designed and constructed based upon high-resolution structures of the corresponding prototype molecules, the Tetrahymena group I self-splicing intron RNA and two proteins (bacteriophage lambdaN and HIV Rev proteins) containing RNA-binding motifs. The splicing reaction proceeds efficiently only when the designed RNA associates with the designed protein either in vivo or in vitro. In vivo mutagenic protein selection was effective for improving the capability of the protein. Kinetic analyses indicate that the protein promotes RNA folding to establish an active conformation. The fact that the conversion of a ribozyme to an RNP can be accomplished by simple molecular design supports the RNA world hypothesis and suggests that a natural active RNP might have evolved readily from a ribozyme.

A comparative study on two GNRA-tetraloop receptors: 11-nt and IC3 motifs.

Natural RNAs often contain terminal loops consisting of GNRA (N=A, G, C, U; R=A, G) and their receptors, which bind to the loops via long-range RNA-RNA interactions. Among several known receptors, two characteristic structural elements have been identified that are termed the 11-nt motif (CCUAAG-UAUGG) and IC3 motif (CCCUAAC-GAGGG). These two motifs that share a similar secondary structure have been shown to exhibit distinctively different binding specificities. The 11-nt motif recognizes a GAAA loop with highest specificity among the known receptors, whereas the IC3 motif distinguishes GAAA from other GNRA loops less stringently than any other receptors. To identify the elements in the receptors that determine the binding specificity, a series of chimeric receptors derived from the two motifs were prepared and their properties were examined. We identified characteristic base-pairs and a particular U residue in the receptors as such elements by means of a gel mobility shift assay that evaluates the degree of the tetraloop-receptor interaction. The relationship between the elements and the specificity is discussed together with a model that describes a possible evolutional linkage between the two receptors.

Conserved base-pairings between C266-A268 and U307-G309 in the P7 of the Tetrahymena ribozyme is nonessential for the in vitro self-splicing reaction.

P7 is highly conserved in Group I self-splicing intron ribozymes. This region is known to coordinate metal ions and bind a cofactor guanosine required for the self-splicing. To further investigate the fundamental role of the corresponding region in the Tetrahymena ribozyme, we attempted to identify minimal requirements for the base-paired region excluding the guanosine binding site. We discovered that a variety of sequences are eligible and its derivatives possessing extra nucleotide(s) can still conduct the first step of splicing, indicating that no particular base-pairing is essential in this region for conducting the reaction in vitro. The results provide two hypotheses for the fundamental role of this region: (i) if the region contains element(s) that are strictly required in the catalysis, they are not necessarily tightly fixed in the ribozyme and (ii) if not, its fundamental role may simply be to coordinate neighboring regions that are directly involved in the catalysis.

A protective role of PKCepsilon against TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in glioma cells.

To elucidate the molecular mechanism(s) involved in the TRAIL-induced apoptosis sensitivity, we conducted the following experiments utilizing TRAIL-sensitive and -resistant glioma cells. We examined the expression of TRAIL receptors mRNA, but no significant differences were detected in those cells. TRAIL-resistant cells were sensitized to TRAIL-induced apoptosis by staurosporine pretreatment and preferentially expressed PKCepsilon. Since several lines of evidence suggest that PKC may play a protective role for apoptosis, we analyzed the involvement of PKCepsilon in TRAIL-induced apoptosis by an adenovirus vector expression system. We found that TRAIL susceptibility was augmented by the expression of a dominant negative PKCepsilon in TRAIL-resistant cells. Conversely, PKCepsilon introduction in TRAIL-sensitive cells resulted in the reduction of TRAIL-induced apoptosis. Taken together, these data suggest that PKCepsilon may be a regulator of susceptibility to TRAIL-induced apoptosis in gliomas and probably other malignancies.

Stem cell factor and interleukin-3 induce stepwise generation of erythroid precursor cells from a basic fibroblast growth factor-dependent hematopoietic stem cell line, A-6.

A multipotent immature myeloid cell population was produced from a basic fibroblast growth factor (bFGF)-dependent hematopoietic stem cell line, A-6, when cultured with stem cell factor (SCF) replacing bFGF. Those cells were positive for stem cell markers, c-kit and CD34, and a myeloid cell marker, F4/80. Some cell fractions were also positive for Mac-1, a macrophage marker or Gr-1, a granulocytic maker, but negative for an erythroid marker TER119. They also showed the expression of mRNA for the myeloid-specific PU.1 but did not that for the erythroid-specific GATA-1. Among various cytokines, interleukin-3 (IL-3) induced erythroid precursor cells that expressed the erythroid-specific GATA-1 and beta-major globin. The quantitative analysis showed that erythroid precursor cells were newly produced from the immature myeloid cells by cultivation with IL-3. SCF and IL-3 induced stepwise generation of erythroid precursor cells from an A-6 hematopoietic stem cell line.

Regulatory domain of protein stability of human P51/TAP63, a P53 homologue.

The amino terminal of human P51/TAp63, a P53 homologue, possesses a transactivation domain involved in the activation of its target genes by binding to DNA elements responsive to the p53 protein family. Using a series of amino terminal deletions, the transactivation domain was mapped between amino acid residues 50 to 69. This domain also regulates protein stability in a proteasome-dependent manner, and Ser51 and Ser68 were found to be essential for this stability. Our results suggest that P51 activity is greatly affected by protein stability.

The transcription factor FoxH1 (FAST) mediates Nodal signaling during anterior-posterior patterning and node formation in the mouse.

FoxH1 (FAST) is a transcription factor that mediates signaling by transforming growth factor-beta, Activin, and Nodal. The role of FoxH1 in development has now been investigated by the generation and analysis of FoxH1-deficient (FoxH1(-/-)) mice. The FoxH1(-/-) embryos showed various patterning defects that recapitulate most of the defects induced by the loss of Nodal signaling. A substantial proportion of FoxH1(-/-) embryos failed to orient the anterior-posterior (A-P) axis correctly, as do mice lacking Cripto, a coreceptor for Nodal. In less severely affected FoxH1(-/-) embryos, A-P polarity was established, but the primitive streak failed to elongate, resulting in the lack of a definitive node and its derivatives. Heterozygosity for nodal renders the FoxH1(-/-) phenotype more severe, indicative of a genetic interaction between FoxH1 and nodal. The expression of FoxH1 in the primitive endoderm rescued the A-P patterning defects, but not the midline defects, of FoxH1(-/-) mice. These results indicate that a Nodal-FoxH1 signaling pathway plays a central role in A-P patterning and node formation in the mouse.

Self-splicing of the Tetrahymena group I ribozyme without conserved base-triples.

BACKGROUND: Group I introns share a conserved core region consisting of two domains, P8-P3-P7 and P4-P6, joined by four base-triples. We showed previously that the T4 td intron can perform phosphoester transfer reactions at two splice sites in the absence of both P4-P6 and the conserved base-triples, whereas it is barely able to perform the intact splicing reaction due to the difficulty of conducting the sequential reactions. RESULTS: Based on previous findings, we constructed a bimolecular ribozyme lacking a large portion of P4-P6 and the base-triples from the Tetrahymena intron, on the assumption that the long-range interactions of the peripheral regions in the two RNAs can compensate for the deteriorated core. The bimolecular ribozyme performed the intact splicing reaction. CONCLUSION: The present analysis indicates that the base-triples are nonessential, but that L4 and the distal part of P4 in P4-P6 are important for conducting the splicing reaction. The reconstituted self-splicing ribozyme provides an amenable system for analysing the role(s) of elements in the core region in the self-splicing reaction mechanism.

A deteriorated triple-helical scaffold accelerates formation of the Tetrahymena ribozyme active structure.

The Tetrahymena group I ribozyme requires a hierarchical folding process to form its correct three-dimensional structure. Ribozyme activity depends on the catalytic core consisting of two domains, P4-P6 and P3-P7, connected by a triple-helical scaffold. The folding proceeds in the following order: (i) fast folding of the P4-P6 domain, (ii) slow folding of the P3-P7 domain, and (iii) structure rearrangement to form the active ribozyme structure. The third step is believed to directly determine the conformation of the active catalytic domain, but as yet the precise mechanisms remain to be elucidated. To investigate the folding kinetics of this step, we analyzed mutant ribozymes having base substitution(s) in the triple-helical scaffold and found that disruption of the scaffold at A105G results in modest slowing of the P3-P7 folding (1.9-fold) and acceleration of step (iii) by 5.9-fold. These results suggest that disruption or destabilization of the scaffold is a normal component in the formation process of the active structure of the wild type ribozyme.

Specific association of a set of molecular chaperones including HSP90 and Cdc37 with MOK, a member of the mitogen-activated protein kinase superfamily.

We have recently identified and cloned a novel member of mitogen-activated protein kinase superfamily protein, MOK (Miyata, Y., Akashi, M., and Nishida, E. (1999) Genes Cells 4, 299-309). To address its regulatory mechanisms, we searched for cellular proteins that specifically associate with MOK by co-immunoprecipitation experiments. Several cellular proteins including a major 90-kDa molecular chaperone HSP90 were found associated with MOK. Treatment of cells with geldanamycin, an HSP90-specific inhibitor, rapidly decreased the protein level of MOK, and the decrease was attributed to enhanced degradation of MOK through proteasome-dependent pathways. Our data suggest that the association with HSP90 may regulate intracellular protein stability and solubility of MOK. Experiments with a series of deletion mutants of MOK indicated that the region encompassing the protein kinase catalytic subdomains I-IV is required for HSP90 binding. Closely related protein kinases (male germ cell-associated kinase and male germ cell-associated kinase-related kinase) were also found to associate with HSP90, whereas conventional mitogen-activated protein kinases (extracellular signal-regulated kinase, p38, and c-Jun N-terminal kinase/stress-activated protein kinase) were not associated with HSP90. In addition, we found that other molecular chaperones including Cdc37, HSC70, HSP70, and HSP60 but not GRP94, FKBP52, or Hop were detected specifically in the MOK-HSP90 immunocomplexes. These results taken together suggest a role of a specific set of molecular chaperones in the stability of signal-transducing protein kinases.

Analysis of molecular interactions of the p53-family p51(p63) gene products in a yeast two-hybrid system: homotypic and heterotypic interactions and association with p53-regulatory factors.

p51 in the p53 tumor suppressor family, also referred to as p63, encodes multiple isoforms including p51A (TAp63gamma) and p51B (TAp63alpha). The p53 protein forms a tetramer, and its stability and activity are regulated by molecular association with viral and cellular proteins and by biochemical modifications. Using a yeast two-hybrid system, the p51A and p51B isoforms were examined for homotypic and heterotypic interactions in the p53 family proteins and for their affinity to the p53-regulatory factors. Results indicate a homotypic interaction dependent on the presumed oligomerization domain of the p51 proteins. The possibility of a weak heterotypic interaction between p51 and p73 proteins was suggested, while association between p51 and p53 appeared improbable. Furthermore, unlike p53, the p51 proteins failed to display an affinity to SV40 large T antigen or MDM2-family proteins. Having several features in common with p53, the p51 proteins may function in biological processes apart from p53.

A simulated molecular evolution from minimal catalytic domain of a group I ribozyme.

Catalysis of Group I intron ribozymes is carried out by its core region consisting of two helical domains P4-P6 and P3-P7. Recently, our laboratory showed that a mutant Group I ribozyme lacking both the P4-P6 domain and the base-triples can perform the trans-esterification reactions. The result demonstrates that the elements required for splicing are concentrated in the P3-P7 domain. Based on this result, we carried out in vitro selection experiment starting from newly constructed libraries in the ribozyme lacking the P4-P6 domain and the base-triples. This selection experiment showed unexpected divergency on the limited sequence space for the active ribozymes.

Minimal catalytic domain of a group I self-splicing intron RNA.

The self-splicing intron ribozymes have been regarded as primitive forms of the splicing machinery for eukaryotic pre-mRNAs. The splicing activity of group I self-splicing introns is dependent on an absolutely conserved and exceptionally densely packed core region composed of two helical domains, P3-P7 and P4-P6, that are connected rigidly via base triples. Here we show that a mutant group I intron ribozyme lacking both the P4-P6 domain and the base triples can perform the phosphoester transfer reactions required for splicing at both the 5' and 3' splice sites, demonstrating that the elements required for splicing are concentrated in the stacked helical P3-P7 domain. This finding establishes that the conserved core of the intron consists of two physically and functionally separable components, and we present a model showing the architecture of a prototype of this class of intron and the course of its molecular evolution.

Structure-function relationships of two closely related group IC3 intron ribozymes from Azoarcus and Synechococcus pre-tRNA.

The two group IC3 pre-tRNA introns from Azoarcus and Synechococcus share very analogous secondary structures. They are small group I ribozymes that possess only two peripheral domains, P2 and P9. However, the 3'-splice site hydrolysis activity of the Synechococcus ribozyme critically depends on P2 whereas that of Azoarcus does not, indicating that the structure-function relationships of the two ribozymes are strikingly different despite their structural resemblance. To identify the element(s) that determines the catalytic properties of these ribozymes, we undertook analyses of chimeric ribozymes prepared by swapping their structural elements. We found that the difference can be attributed to a small number of nucleotides within the conserved core region. Further analysis by employing in vitro selection revealed that a base triple interaction (P4bp3 x J6/7-2) is a critical element for determining activity and suggests the existence of a novel base quintuple involving the base triple P4bp5 x J8/7-5.