Msx2 deficiency in mice causes pleiotropic defects in bone growth and ectodermal organ formation.

The composite structure of the mammalian skull, which forms predominantly via intramembranous ossification, requires precise pre- and post-natal growth regulation of individual calvarial elements. Disturbances of this process frequently cause severe clinical manifestations in humans. Enhanced DNA binding by a mutant MSX2 homeodomain results in a gain of function and produces craniosynostosis in humans. Here we show that Msx2-deficient mice have defects of skull ossification and persistent calvarial foramen. This phenotype results from defective proliferation of osteoprogenitors at the osteogenic front during calvarial morphogenesis, and closely resembles that associated with human MSX2 haploinsufficiency in parietal foramina (PFM). Msx2-/- mice also have defects in endochondral bone formation. In the axial and appendicular skeleton, post-natal deficits in Pth/Pthrp receptor (Pthr) signalling and in expression of marker genes for bone differentiation indicate that Msx2 is required for both chondrogenesis and osteogenesis. Consistent with phenotypes associated with PFM, Msx2-mutant mice also display defective tooth, hair follicle and mammary gland development, and seizures, the latter accompanied by abnormal development of the cerebellum. Most Msx2-mutant phenotypes, including calvarial defects, are enhanced by genetic combination with Msx1 loss of function, indicating that Msx gene dosage can modify expression of the PFM phenotype. Our results provide a developmental basis for PFM and demonstrate that Msx2 is essential at multiple sites during organogenesis.

Use of TLR9 and TLR7/8 agonists in combination with d-galactosamine in exploring models for distinct severities of systemic inflammation relative to liver injury.

Challenges with various TLR ligands (TLRLs)in combination with D-galactosamine (GalN) in rodents may mimic diverse conditions of acute inflammation and organ failure. Here, we report that CpG (ODN1826, TLR9 agonist)/GalN induced a liver-specific injury with modest systemic effects, whereas R848 (resiquimod, TLR7/8 agonist)/GalN exhibited systemic and liver toxicity. We also observed the protective effect of Gr-1+ cells (the population containing neutrophils) against liver injury in both the R848/GalN and CpG/GalN models. In cytokine measurements, the intraperitoneal administration of antibodies showed a non-specific tolerance induction effect, which was more pronounced in the CpG/GalN than in the R848/GalN model. Cytokine analyses also suggested that the TLR9 agonist/GalN induced a limited degree of systemic inflammation compared to TLR7/8 agonist/GalN models. The relevance of this finding to the TLR9-mediated induction of stress tolerance (protective effect) in non-immune cells is discussed.

Microtubule-organizing centers abnormal in number, structure, and nucleating activity in x-irradiated mammalian cells.

Microtubule-organizing centers (MTOCs) in x-irradiated cells were visualized by immunofluorescence using antibody against tubulin. From two to ten reassembly sites of microtubules appeared after microtubule depolymerization at low temperature in an irradiated mitotic cell, in contrast to nonirradiated mitotic cells, which predominantly show 2 MTOCs. A time-course examination of MTOCs in synchronously cultured cells revealed that the multiple MTOCs appeared not immediately after irradiation but at the time of mitosis. Those multiple MTOCs formed at mitosis were inherited by the daughter cells in the next generation. The structure and capacity of the centrosomes to nucleate microtubules in vitro were then examined by electron microscopy of whole-mount preparations as well as by dark-field microscopy. About 70-80% of the centrosomes derived from nonirradiated cells were composed of a pair of centrioles and pericentriolar material, which initiated greater than 100 microtubules. The fraction of fully active complete centrosomes decreased with time of incubation after irradiation. These were replaced by disintegrated centrosomal components such as dissociated centrioles and pericentriolar cloud, a nucleating site with a single centriole, or only an amorphous structure of pericentriolar cloud. Assembly of less than 20 microtubules onto the amorphous cloud without centrioles was seen in 54% of the initiating sites in mitotic cells 2 d after irradiation. These results suggest that x-irradiation causes disintegration of centrosomes at mitosis when the structural and functional reorganization of centrosomes is believed to occur.

Effect upon mitogenic stimulation of calcium-dependent phosphorylation of cytoskeleton-associated 350,000- and 80,000-mol-wt polypeptides in quiescent 3Y1 cells.

Rabbit antiserum raised against highest molecular weight microtubule-associated protein (MAP-1) of brain immunoprecipitated 350,000-, 300,000-, and 80,000-mol-wt phosphoproteins of rat embryo fibroblasts (3Y1-B). The 350,000-mol-wt protein was sensitive to heat as was brain MAP-1, but the 300,000- and 80,000-mol-wt proteins were not. These polypeptides were hardly phosphorylated in cells in the quiescent G0 phase but were rapidly phosphorylated after addition of serum, epidermal growth factor, phorbol ester, insulin, or transferrin in the presence of calcium ions. All these agents also induced incorporation of [3H]-thymidine into DNA. These polypeptides were detected in isolated microtubules and cold-resistant filaments by immunoblotting. Since the 350,000-mol-wt polypeptide was detected in the membrane, the cytoskeletons, and the nucleus, and has been suggested to function as a linker, its rapid phosphorylation might represent an early process in transduction of the signal of mitogenic stimulation to the nucleus.

Surface dose measurement in patients and physicians and effective dose estimation in patients during uterine artery embolisation.

Surface dose monitoring in patients and physicians during 29 uterine artery embolisation (UAE) procedures was performed using photoluminescence dosemeters and thermo-luminescence dosemeters. Organ or tissue doses were measured with an anthropomorphic phantom using UAE exposure conditions averaged from the 29 cases, and effective doses were estimated for the patient. Entrance surface dose of the patients at the maximum dose position ranged from 121.5 to 1650 mGy. Estimated doses ranged from 3.16 to 43 mGy for the ovary and from 3.8 to 51.8 mGy for the uterus. The effective dose was 1.09-14.8 mSv. Monitored doses on the body surface of physicians were relatively high in the upper arm (5.41+/-1.52 to 163+/-17.25 microGy) and the hand and fingers (0.85+/-1.18 to 222+/-16.4 microGy).

Phosphorylation of native and reassembled neurofilaments composed of NF-L, NF-M, and NF-H by the catalytic subunit of cAMP-dependent protein kinase.

Phosphorylation of neurofilament-L protein (NF-L) by the catalytic subunit of cAMP-dependent protein kinase (A-kinase) inhibits the reassembly of NF-L and disassembles filamentous NF-L. The effects of phosphorylation by A-kinase on native neurofilaments (NF) composed of three distinct subunits: NF-L, NF-M, and NF-H, however, have not yet been described. In this paper, we examined the effects of phosphorylation of NF proteins by A-kinase on both native and reassembled filaments containing all three NF subunits. In the native NF, A-kinase phosphorylated each NF subunit with stoichiometries of 4 mol/mol for NF-L, 6 mol/mol for NF-M, and 4 mol/mol for NF-H. The extent of NF-L phosphorylation in the native NF was nearly the same as that of purified NF-L. However, phosphorylation did not cause the native NFs to disassemble into oligomers, as was the case for purified NF-L. Instead, partial fragmentation was detected in sedimentation experiments and by electron microscopic observations. This is probably not due to the presence of the three NF subunits in NF or to differences in phosphorylation sites because reassembled NF containing all three NF subunits were disassembled into oligomeric forms by phosphorylation with A-kinase and the phosphorylation by A-kinase occurred at the head domain of NF-L whether NF were native or reassembled. Disassembling intermediates of reassembled NF containing all three NF subunits were somewhat different from disassembling intermediates of NF-L. Thinning and loosening of filaments was frequently observed preceding complete disassembly. From the fact that the thinning was also observed in the native filaments phosphorylated by A-kinase, it is reasonable to propose the native NF is fragmented through a process of thinning that is stimulated by phosphorylation in the head domain of the NF subunits.

Development of a portable system for checking radioactive sources using long wave radio frequency identification.

A portable system for automatically checking radioactive sources stored in lead containers at low temperatures was developed in order to prevent the discharging of orphan sources and contaminated materials from a controlled area to the general public. A radio frequency identification (RFID) system using a long wave in a frequency range of 125 kHz was composed of identification tags, a reader, a notebook computer, and software. ID tags without batteries were devised by using integrated circuits with an electrically erasable programmable read-only memory of 250 bytes and antennas. This software consisted of operating and maintenance functions. The read range of the ID tags was adjusted to around 5 cm in order to avoid accidental contamination and for discriminating the multiple sources. A water layer of 6.9 cm had no influence on communication between the ID tags and the reader. The data of the ID tags stored at +4, -20, and -80 degrees C were precisely read 4 mo later. The influence of lead was completely removed by separating the ID tags more than 1.6 cm from the lead. A reader can exactly identify the data of the ID tags within 6.0 cm at a velocity less than 9.0 cm s(-1). Performance of the software was verified using mock data. Nine lists concerning registered, disposed, and missing sources, etc., were displayed on the computer monitor and printed out. An RFID system using long waves proved to be applicable for routinely checking radioactive sources.

Comparison of patient doses in 256-slice CT and 16-slice CT scanners.

The 256-slice CT-scanner has been developed at the National Institute of Radiological Sciences. Nominal beam width was 128 mm in the longitudinal direction. When scanning continuously at the same position to obtain four-dimensional (4D) images, the effective dose is increased in proportion to the scan time. Our purpose in this work was to measure the dose for the 256-slice CT, to compare it with that of the 16-slice CT-scanner, and to make a preliminary assessment of dose for dynamic 3D imaging (volumetric cine imaging). Our group reported previously that the phantom length and integration range for dosimetry needed to be at least 300 mm to represent more than 90% of the line integral dose with the beam width between 20 mm and 138 mm. In order to obtain good estimates of the dose, we measured the line-integral dose over a 300 mm range in PMMA (polymethylmethacrylate) phantoms of 160 mm or 320 mm diameter and 300 mm length. Doses for both CT systems were compared for a clinical protocol. The results showed that the 256-slice CT generates a smaller dose than the 16-slice CT in all examinations. For volumetric cine imaging, we found an acceptable scan time would be 6 s to 11 s, depending on examinations, if dose must be limited to the same values as routine examinations with a conventional multidetector CT. Finally, we discussed the studies necessary to make full use of volumetric cine imaging.

Amino acid and nucleotide recurrence in aligned sequences: synonymous substitution patterns in association with global and local base compositions.

The tendency for repetitiveness of nucleotides in DNA sequences has been reported for a variety of organisms. We show that the tendency for repetitive use of amino acids is widespread and is observed even for segments conserved between human and Drosophila melanogaster at the level of >50% amino acid identity. This indicates that repetitiveness influences not only the weakly constrained segments but also those sequence segments conserved among phyla. Not only glutamine (Q) but also many of the 20 amino acids show a comparable level of repetitiveness. Repetitiveness in bases at codon position 3 is stronger for human than for D.melanogaster, whereas local repetitiveness in intron sequences is similar between the two organisms. While genes for immune system-specific proteins, but not ancient human genes (i.e. human homologs of Escherichia coli genes), have repetitiveness at codon bases 1 and 2, repetitiveness at codon base 3 for these groups is similar, suggesting that the human genome has at least two mechanisms generating local repetitiveness. Neither amino acid nor nucleotide repetitiveness is observed beyond the exon boundary, denying the possibility that such repetitiveness could mainly stem from natural selection on mRNA or protein sequences. Analyses of mammalian sequence alignments show that while the 'between gene' GC content heterogeneity, which is linked to 'isochores', is a principal factor associated with the bias in substitution patterns in human, 'within gene' heterogeneity in nucleotide composition is also associated with such bias on a more local scale. The relationship amongst the various types of repetitiveness is discussed.

Early decrease in hyaluronidase-sensitive cell surface charge during the differentiation of Friend erythroleukemic cells by dimethyl sulfoxide.

Early membrane events in erythroid differentiation were investigated by means of cell electrophoresis utilizing cultured Friend erythroleukemia cell clones of different inducibility. The cell electrophoretic mobility decreased by 18% within 30 min of treatment with 1.5% dimethyl sulfoxide (DMSO) in highly inducible clones but not in noninducible clones. The reduced mobility persisted for 5 days of incubation with DMSO until hemoglobin synthesis. DMSO treatment for less than 16 hr and subsequent incubation without the drug resulted in the complete recovery of the mobility and no hemoglobin synthesis. Longer exposure to DMSO resulted in the loss of recovery of mobility and an increasing fraction of benzidine-positive cells seen on Day 5. Measurement of the electrophoretic mobility after the removal of acidic sugars by their specific enzymes suggested that hyaluronidase-sensitive negative charges were lost from the cell surface only in highly inducible clones. The mobility reduction associated with hyaluronic acid was also caused by other potent inducers (sodium butyrate, N-methylacetamide, and N,N-dimethylacetamide). These results suggest that the decrease in cell surface glycocalyx might be an early step in the induction of differentiation of Friend erythroleukemia cells.

Translocation of hyaluronic acid in cell surface of cultured mammalian cells after x-irradiation and its recovery by added adenosine triphosphate.

To investigate the mechanism of radiation-induced decrease in cell electrophoretic mobility and its recovery by added adenosine triphosphate, specific enzymes and buffer solutions of different ionic strength were utilized. Decrease in the mobility of irradiated cells was detected only with the buffer solution of ionic strengths higher than 0.100. In this range of ionic strengths, removal of hyaluronic acid from cell surface by hyaluronidase had no effect on the electrophoretic mobility of irradiated cells, while the enzyme treatment resulted in 27% mobility reduction in non-irradiated cells. The removal of sialic acid and chondroitin sulfate by their specific enzymes resulted in the similar decrease in mobility either in irradiated and non-irradiated cells. These results suggest that the X-ray induced translocation of hyaluronic acid from the peripheral zone of O--7.5 A into the deeper zone of about 10--17 A, if we use the Debye-Hückel's thickness of ion atmosphere for an approximate estimate of effective depth of electrokinetic plane of shear. Hyaluronic acid reappeared to the peripheral zone by the subsequent incubation after small dose irradiation, or by the addition of 1 mM adenosine triphosphate with Ca2+.

Small amount of concanavalin A modifies radiation-induced alteration in cell-surface charge depending on its binding condition.

Cell electrophoretic mobility of cultured melanoma cells or rat erythrocytes decreased with time after X-irradiation. Addition of tetravalent concanavalin A or divalent succinyl-concanavalin A before (not after) irradiation, completely blocked the mobility reduction in greater concentrations than 5 mug/l. At 5 mug/l only 3.7 - 10(3) concanavalin A molecules bound to receptors per cell, while 4.18 - 10(7) molecules/cell bound at saturating concentrations. Preincubation with concanavalin A at 37 degrees C was effective even when the cells were treated with alpha-methylmannoside immediately after irradiation. At low temperature, however, concanavalin A was not effective despite a sufficient amount of bound 125I-labelled concanavalin A. Treatment with alpha-methylmannoside following the binding of concanavalin A at 37 degrees C before irradiation inhibited the concanavalin A effect depending on temperature. The residual amount of bound lectin could not account for the temperature dependence. The amount of sialic acid (the main charged substance) was not altered by X-irradiation with or without the lectin. Divalent succinyl-concanavalin A was also effective in blocking the radiation effect on electrophoretic mobility. These results seem to suggest that binding of a very small amount of concanavalin A without causing cell agglutination or clustering of its receptors, induces some alteration in the conformation of receptor glycoprotein, which blocks the internalization of acidic sugar residues by subsequent irradiation.

Tendency for local repetitiveness in amino acid usages in modern proteins.

Systematic analyses of human proteins show that neural and immune system-specific, and therefore, relatively "modern" proteins have a tendency for repetitive use of amino acids at a local scale ( approximately 1-20 residues), while ancient proteins (human homologues of Escherichia coli proteins) do not. Those protein subsegments which are unique based on homology search account for the repetitiveness. Simulation shows that such repetitiveness can be maintained by frequent duplication on a very short scale (one to two codons) in the presence of substitutive point mutation, while the latter tends to mitigate the repetitiveness. DNA analyses also show the presence of cryptic (i.e. "out of the codon frame") repetitiveness, which cannot fully be explained by features in protein sequences. Simulative modification of the amino acid sequences of immune system-specific proteins estimate that 2.4 duplication events occur during the period equivalent to ten events of substitution mutation. It is also suggested that the repetitiveness leads to longitudinal unevenness within a given peptide domain. Those peptide motifs which contain similarly charged residues are likely to be generated more frequently in the presence of the tendency for repetitiveness than in its absence. Therefore, the neutral propensity of DNA for duplication, which can also tend to generate repetitiveness in amino acid sequences, seems to be manifested primarily when the constraints on amino acid sequences are relatively weak, and yet may be positively contributing to generation of unevenness in modern proteins.

Genetic analysis of rolled, which encodes a Drosophila mitogen-activated protein kinase.

Genetic and molecular characterization of the dominant suppressors of D-raf(C110) on the second chromosome identified two gain-of-function alleles of rolled (rl), which encodes a mitogen-activated protein (MAP) kinase in Drosophila. One of the alleles, rl(Su23), was found to bear the same molecular lesion as rl(Sem), which has been reported to be dominant female sterile. However, rl(Su23) and the current stock of rl(Sem) showed only a weak dominant female sterility. Detailed analyses of the rl mutations demonstrated moderate dominant activities of these alleles in the Torso (Tor) signaling pathway, which explains the weak dominant female sterility observed in this study. The dominant rl mutations failed to suppress the terminal class maternal-effect mutations, suggesting that activation of Rl is essential, but not sufficient, for Tor signaling. Involvement of rl in cell proliferation was also demonstrated by clonal analysis. Branching and integration of signals in the MAP kinase cascade is discussed.

Genetic instability of p53-deficient mouse cells.

We established fibroblast cultures from p53-deficient mouse embryos, which were originally constructed by Dr. S. Aizawa (Kumamoto Univ.). These p53-deficient fibroblasts showed the same sensitivity to UV and X-ray as wild-type fibroblasts. There was no difference between repair activity of UV-induced DNA damages in p53-deficient and wild-type cells, either. However, UV-induced sister chromatid exchanges were significantly increased and delay of entering S-phase after UV-irradiation was reduced in p53-deficient cells indicating abnormality in the checkpoint function of the cell cycle in p53-deficient cells. We also used the supF gene on a shuttle vector to analyze UV-induced mutations in p53-deficient cells. Although frequencies of UV-induced mutations were not different between p53-deficient and wild-type cells, distributions of base-substitution mutations on the supF gene were different.

Blood flow of human soft tissue sarcomas measured by thallium-201 scanning: prediction of tumor response to radiation.

Thallium-201 chloride (201T1) has been used to determine regional perfusion in the myocardium and in tumors. This study was done to determine the potential prognostic importance of lesion tracer uptake to regression, local control, and rate of distant metastasis in 14 patients with neoplasms of soft tissue. Most patients had planned resections following preoperative radiation therapy. Minimum follow-up was 4 years. The ratio of nuclide uptake in the tumor to surrounding normal tissue was used as an estimate of relative blood flow. Tumors with acute volume responses (greater than or equal to 50% at the completion of X irradiation) had lower 201T1 uptake indicating lower relative blood flow than tumors that failed to have a volume reduction [1.63 +/- 0.30 (n = 9) vs 3.49 +/- 0.41 (n = 5) 201T1]. All patients had local tumor control. Patients with high uptake tumors tended to develop metastases at a higher frequency, although this was not statistically significant (p = 0.10). We conclude that 201T1 scans are a safe, non-invasive method of estimating tumor perfusion which can be useful to predict acute response to radiation, and may help to identify patients who will ultimately develop distant metastases.

Association of neonatal hyperbilirubinemia with bilirubin UDP-glucuronosyltransferase polymorphism.

OBJECTIVE: The incidence of nonphysiologic neonatal hyperbilirubinemia is twice as high in East Asians as in whites. We studied whether the condition was associated with mutations in the gene for bilirubin uridine 5'-diphosphate-glucuronosyltransferase (UGT1A1), a key enzyme of bilirubin catabolism. DESIGN: We analyzed the UGT1A1 gene in 25 Japanese neonates who had nonphysiologic hyperbilirubinemia (serum bilirubin >257 micromol/L) with no obvious cause. They had all received phototherapy. The background control population consisted of 50 Japanese neonates whose transcutaneous jaundice index was monitored during the first week of life. We detected mutations by direct sequencing of polymerase chain reaction-amplified fragments of the gene. RESULTS: We found a polymorphism for UGT1A1 in exon 1; a G-->A transition at nucleotide 211 caused arginine to replace glycine at position 71 of corresponding protein product (G71R). The frequency of the mutated allele in the hyperbilirubinemic group (0.34) was significantly higher (chi2 = 5.56) than in the control group (0.16). In the control group the peak transcutaneous jaundice index of the carriers of G71R was significantly higher than it was in the normal infants. CONCLUSIONS: The missense mutation causing G71R is the first reported polymorphism for UGT1A1, and the mutation is a risk factor for nonphysiologic neonatal hyperbilirubinemia. The high incidence of hyperbilirubinemia in the Japanese may be attributable to the high frequency of this missense mutation.

Porous calcium phosphate coating over phosphorylated chitosan film by a biomimetic method.

A porous calcium phosphate coating deposited on chitosan films was studied using scanning electron microscopy, energy-dispersive X-ray analysis, micro-Fourier transform infrared spectroscopy (micro-FTIR) and thin-film X-ray diffractometry (XRD). Chitosan films were first prepared by dissolving chitosan powder in dilute acetic acid and drying in a flat petri dish. The films were phosphorylated using urea and H3PO4 with the P content being 0.1-0.2 wt%. Phosphorylated films soaked in saturated Ca(OH)2 solution for 8 days led to the formation of a calcium phosphate precursor phase over the entire surface. This precursor phase stimulated the growth of a porous coating of calcium-deficient hydroxy apatite when immersed in 1.5 x SBF for more than 20 days. Phosphorylated films not treated with Ca(OH)2 did not show any calcium phosphate growth upon immersion in SBF solution. The precursor phase is thought to be octacalcium phosphate, which nucleates a HAP phase during SBF treatment. Initially, this treatment in SBF results in the formation of a single-layer calcium phosphate particles over the film surface. As immersion time in SBF increases, further nucleation and growth produce a porous HAP coating. The Ca/P ratio of the HAP coating is a function of SBF immersion time.

The trimannosyl cores of N-glycans are important for the procoagulant protease-inhibitory activity of urinary protein C inhibitor.

We investigated the relationship between the procoagulant protease-inhibitory activity and the N-glycan structures in urinary protein C inhibitor (uPCI) by sequential exoglycosidase digestions based on the N-glycan structures elucidated in this report. uPCI was glycosylated on the three potential N-glycosylation sites, asparagines 230, 243 and 319 (N230, N243 and N319) in the molecule and had four biantennary complex type sugar chains. The inhibitory activities of uPCI toward thrombin and plasma kallikrein were little changed by the sequential removal of N-acetylneuraminic acid and galactose residues from the termini and N-acetylglucosamine residues from the branches of the N-glycans. However, the inhibitory activities were markedly decreased by further removing alpha-mannose residues from the trimannosyl cores of the N-glycans. These results suggest that the trimannosyl cores of N-glycans are important for uPCI to inhibit the procoagulant protease.

Alignment of x-ray tube focal spots for spectral measurement.

A general method to align a diagnostic x-ray machine for x-ray spectrum measurement purpose was theoretically and experimentally investigated by means of the optical alignment of focal pinhole images. Focal pinhole images were obtained by using a multi-pinholed lead plate. the vertical plane, including the central axis and tube axis, was decided upon by observing the symmetry of focal images. the central axis was designated as a line through the center of focus parallel to the target surface lying in the vertical plane. A method to determine the manipulation of the central axis in any direction is presented.