Gli2 protein expression level is a feasible marker of ligand-dependent hedgehog activation in pancreatic neoplasms.

The hedgehog pathway is known to promote proliferation of pancreatic ductal adenocarcinoma (PDA) and has been shown to restrain tumor progression. To understand how hedgehog causes these effects, we sought to carefully examine protein expression of hedgehog signaling components during different tumor stages. Genetically engineered mice, Pdx1-Cre;LSL-KrasG12D and Pdx1-Cre;LSL-KrasG12D;p53lox/+, were utilized to model distinct phases of tumorigenesis, pancreatic intraepithelial neoplasm (PanIN) and PDA. Human pancreatic specimens of intraductal papillary mucinous neoplasm (IPMN) and PDA were also employed. PanIN and IPMN lesions highly express Sonic Hedgehog, at a level that is slightly higher than that observed in PDA. GLI2 protein is also expressed in both PanIN/IPMN and PDA. Although there was no difference in the nuclear staining, the cytoplasmic GLI2 level in PDA was modest in comparison to that in PanIN/IPMN. Hedgehog interacting protein was strongly expressed in the precursors, whereas the level in PDA was significantly attenuated. There were no differences in expression of Patched1 at early and late stages. Finally, a strong correlation between Sonic Hedgehog and GLI2 staining was found in both human and murine pancreatic tumors. The results indicate that the GLI2 protein level could serve as a feasible marker of ligand-dependent hedgehog activation in pancreatic neoplasms.

Electron and Ion Heating Characteristics during Magnetic Reconnection in the MAST Spherical Tokamak.

Electron and ion heating characteristics during merging reconnection start-up on the MAST spherical tokamak have been revealed in detail using a 130 channel yttrium aluminum garnet (YAG) and a 300 channel Ruby-Thomson scattering system and a new 32 chord ion Doppler tomography diagnostic. Detailed 2D profile measurements of electron and ion temperature together with electron density have been achieved for the first time and it is found that electron temperature forms a highly localized hot spot at the X point and ion temperature globally increases downstream. For the push merging experiment when the guide field is more than 3 times the reconnecting field, a thick layer of a closed flux surface form by the reconnected field sustains the temperature profile for longer than the electron and ion energy relaxation time ~4-10 ms, both characteristic profiles finally forming a triple peak structure at the X point and downstream. An increase in the toroidal guide field results in a more peaked electron temperature profile at the X point, and also produces higher ion temperatures at this point, but the ion temperature profile in the downstream region is unaffected.

The gene encoding a newly discovered protein, chorein, is mutated in chorea-acanthocytosis.

Chorea-acanthocytosis is a neurodegenerative disorder with peripheral red cell acanthocytosis. Linkage of chorea-acanthocytosis to chromosome 9q21 has been found. We refined the locus region and identified a previously unknown, full-length cDNA encoding a presumably structural protein, which we called chorein. We found a deletion in the coding region of the cDNA leading to a frame shift resulting in the production of a truncated protein in both alleles of patients and in single alleles of obligate carriers.

Ion and electron heating characteristics of magnetic reconnection in a two flux loop merging experiment.

Characteristics of the high-power reconnection heating were measured for the first time directly by two-dimensional measurements of ion and electron temperatures. While electrons are heated mainly inside the current sheet by the Ohmic heating power, ions are heated mainly by fast shock or viscosity damping of the reconnection outflow in the two downstream areas. The magnetic reconnection converts the energy of reconnecting magnetic field B(p) mostly to the ion thermal energy, indicating that the reconnection heating energy is proportional to B(p)(2).

Neural substrates of intermanual transfer of a newly acquired motor skill.

In healthy humans, the two cerebral hemispheres show functional specialization to a degree unmatched in other animals, and such strong hemispheric specialization contributes to unimanual skill acquisition [1, 2]. When most humans learn a new motor skill with one hand, this process results in performance improvements in the opposite hand as well [3-6]. Despite the obvious adaptive advantage of such intermanual transfer, there is no direct evidence identifying the neural substrates of this form of skill acquisition [7-9]. Here, we used functional magnetic resonance imaging (fMRI) to study brain regions activated during intermanual transfer of a learned sequence of finger movements. First, we found that the supplementary motor area (SMA) has more activity when a skill has transferred well than when it has transferred poorly. Second, we found that fMRI activity in the ventrolateral posterior thalamic nucleus correlated with successful future intermanual transfer, whereas activity in the ventrolateral anterior thalamic nucleus correlated with past intermanual transfer. Third, we found that repetitive transcranial magnetic stimulation applied over the SMA blocked intermanual transfer without affecting skill acquisition. These findings provide direct evidence for an SMA-based mechanism that supports intermanual transfer of motor-skill learning.

[Experience of chest wall reconstruction with newly developed material].

After the chest wall resection, its reconstruction is often needed. A 45-year-old male lung adenocarcinoma patient with chest wall invasion underwent upper lobectomy of the right lung with partial resection of 4-6th ribs. The size of the removed chest wall was 11 x 6.5 cm. We reconstructed the chest wall with Bard Composix E/X Mesh. This prosthesis is consisted of a polypropylene mesh and an expanded polytetrafluoroethylene sheet This material is seems to be useful in the reconstruction of chest wall in both preventing pulmonary adhesion and enabling good wound healing.

K-ras mutations and cell kinetics in Helicobacter pylori associated gastric intestinal metaplasia: a comparison before and after eradication in patients with chronic gastritis and gastric cancer.

BACKGROUND: Helicobacter pylori related gastric intestinal metaplasia (IM) is considered to be a precancerous lesion. AIMS: To identify the effects of H pylori eradication on K-ras mutations, cell kinetics in IM and histological changes in patients with and without gastric cancers in a one-year prospective study. METHODS: Patients included group A (n = 39), chronic gastritis, and group B (n = 53), intestinal-type early gastric cancer patients who had all undergone endoscopic mucosal resection (n = 25) or surgical resection (n = 28). K-ras codon 12 mutations in IM were examined, followed by DNA sequencing analysis. Proliferating and apoptotic cells were detected with anti-Ki-67 antibody and using the TUNEL method, respectively. RESULTS: The incidence of K-ras mutations in the cancer was only 3.8%. The mutant K-ras in IM was observed more frequently in group A (46.2%) than in group B patients (1.9%) (p<0.005). After eradication, the K-ras mutations significantly declined to 12.8% in group A (p<0.005). The mutation pattern of K-ras codon 12 before eradication was that GGT was mainly changed to AGT (50%) in group A. AGT transformation was not affected by treatment. Apoptosis in IM showed an increase after H pylori eradication in both groups (p<0.05 in group A) although no histological improvement in IM was observed. The monocyte score was significantly higher in group A than in group B (p<0.05); the score improved significantly after eradication. CONCLUSIONS: K-ras mutations in IM do not always play a role in gastric carcinogenesis but cell kinetics, especially apoptosis, in IM may contribute to it. There are early events in K-ras mutations which are influenced by H pylori infection; some mutations may also be selected by eradication. These unstable K-ras mutations in IM may be related to lymphocyte infiltration caused by H pylori infection.

Bloom helicase is involved in DNA surveillance in early S phase in vertebrate cells.

Bloom syndrome (BS) is a recessive human genetic disorder characterized by short stature, immunodeficiency and an elevated risk of malignancy. The gene mutated in BS, BLM, encodes a RecQ-type DNA helicase. BS cells have mutator phenotypes such as hyper-recombination, chromosome instability and an increased frequency of sister chromatid exchange (SCE). To define the primary role of BLM, we generated BLM(-/-) mutants of the chicken B-cell line DT40. In addition to characteristics of BLM(-/-) cells reported previously by the other group, they are hypersensitive to genotoxic agents such as etoposide, bleomycin and 4-nitroquinoline-1-oxide and irradiation with the short wave length of UV (UVC) light, whereas they exhibit normal sensitivity to X-ray irradiation and hydroxyurea. UVC irradiation to BLM(-/-) cells during G(1) to early S phase caused chromosomal instability such as chromatid breaks and chromosomal quadriradials, leading to eventual cell death. These results suggest that BLM is involved in surveillance of base abnormalities in genomic DNA that may be encountered by replication forks in early S phase. Such surveillance would maintain genomic stability in vertebrate cells, resulting in the prevention of cellular tumorigenesis.

Chemical modification of cysteinyl, lysyl and histidyl residues of mouse liver 17 beta-hydroxysteroid dehydrogenase.

Monomeric 17 beta-hydroxysteroid dehydrogenase from mouse liver was rapidly inactivated by 5,5'-dithiobis(2-nitrobenzoic acid) and 2,4,6-trinitrobenzene-1-sulfonate, and the absorption spectra of the inactivated enzymes indicated that cysteine and lysine residues were modified. The kinetics of inactivation and spectrophotometric quantification of the modified residues suggested that complete inactivation was caused by modification of two cysteine residues or one lysine residue per active site. The inactivation by the two reagents was protected by NADP+ and some coenzyme analogs, but not by a steroid substrate, testosterone. Moreover, chemical modification by diethyl pyrocarbonate also produced inactivation of the enzyme, and showed a difference spectrum with a peak at 242 nm characteristic of N-carbethoxyhistidine residues, which decreased with the addition of hydroxylamine. The inactivation by this reagent, following pseudo-first-order kinetics, was protected partially by either NADP+ or testosterone and completely in the presence of both the coenzyme and substrate. The results suggest the presence of essential cysteine and lysine residues at or near the coenzyme-binding site and that of essential histidine residue(s) in the catalytic region of the active site of mouse liver 17 beta-hydroxysteroid dehydrogenase.

Changes of lysosomal proteinase activities and their expression in rat cultured keratinocytes during differentiation.

The cathepsins B, H and L, lysosomal cysteine proteinases, play a major role in intracellular protein degradation. These proteinase activities and expressions were examined in a Ca2+ regulated epidermal culture system which consists of two morphological cell types: undifferentiated cells grown in low Ca2+ (0.1 mM concentration) and differentiated cells grown in high Ca2+ (1.8 mM concentration), respectively. Cathepsin B and L activities of the differentiated cells showed a several-fold increase compared to that of the undifferentiated cells. In addition, by using CM-cellulose column chromatography, cathepsin B and L were separated and the level of cathepsin L activity increased significantly. Cathepsin B, L and H were also detected by using an immunoblotting procedure in which their bands were expressed after differentiation was induced by the increasing calcium concentration. Cathepsin L activity and immunostaining intensity reached a maximum at 1 or 2 days of differentiation. In contrast, cystatin alpha (an endogenous inhibitor of cysteine-dependent cathepsins) appeared in the final stage of differentiation. These results indicate that the expression of epidermal cathepsins and their endogenous inhibitor are involved in part of the program of cell differentiation and the terminal differentiation process in cultured rat keratinocytes.

Chemo-enzymatic synthesis of a bioactive peptide containing a glutamine-linked oligosaccharide and its characterization.

A bioactive peptide containing a glutamine-linked oligosaccharide was chemo-enzymatically synthesized by use of the solid-phase method of peptide synthesis and the transglycosylation activity of endo-beta-N-acetylglucosaminidase. Substance P, a neuropeptide, is an undecapeptide containing two L-glutamine residues. A substance P derivative with an N-acetyl-D-glucosamine residue attached to the fifth or sixth L-glutamine residue from the N-terminal region was chemically synthesized. A sialo complex-type oligosaccharide derived from a glycopeptide of hen egg yolk was added to the N-acetyl-D-glucosamine moiety of the substance P derivative using the transglycosylation activity of endo-beta-N-acetylglucosaminidase from Mucor hiemalis, and a substance P derivative with a sialo complex-type oligosaccharide attached to the L-glutamine residue was synthesized. This glycosylated substance P was biologically active, although the activity was rather low, and stable against peptidase digestion. The oligosaccharide moiety attached to the L-glutamine residue of the peptide was not liberated by peptide-N(4)-(N-acetyl-beta-D-glucosaminyl) asparagine amidase F.

Design of a new surgical approach for ventricular remodeling to relieve ischemic mitral regurgitation: insights from 3-dimensional echocardiography.

BACKGROUND: Mechanistic insights from 3D echocardiography (echo) can guide therapy. In particular, ischemic mitral regurgitation (MR) is difficult to repair, often persisting despite annular reduction. We hypothesized that (1) in a chronic infarct model of progressive MR, regurgitation parallels 3D changes in the geometry of mitral leaflet attachments, causing increased leaflet tethering and restricting closure; therefore, (2) MR can be reduced by restoring tethering geometry toward normal, using a new ventricular remodeling approach based on 3D echo findings. METHODS AND RESULTS: We studied 10 sheep by 3D echo just after circumflex marginal ligation and 8 weeks later. MR, at first absent, became moderate as the left ventricle (LV) dilated and the papillary muscles shifted posteriorly and mediolaterally, increasing the leaflet tethering distance from papillary muscle tips to the anterior mitral annulus (P<0.0001). To counteract these shifts, the LV was remodeled by plication of the infarct region to reduce myocardial bulging, without muscle excision or cardiopulmonary bypass. Immediately and up to 2 months after plication, MR was reduced to trace-to-mild as tethering distance was decreased (P<0.0001). LV ejection fraction, global LV end-systolic volume, and mitral annular area were relatively unchanged. By multiple regression, the only independent predictor of MR was tethering distance (r(2)=0.81). CONCLUSIONS: Ischemic MR in this model relates strongly to changes in 3D mitral leaflet attachment geometry. These insights from quantitative 3D echo allowed us to design an effective LV remodeling approach to reduce MR by relieving tethering.

Mechanism of ischemic mitral regurgitation with segmental left ventricular dysfunction: three-dimensional echocardiographic studies in models of acute and chronic progressive regurgitation.

OBJECTIVES: This study aimed to separate proposed mechanisms for segmental ischemic mitral regurgitation (MR), including left ventricular (LV) dysfunction versus geometric distortion by LV dilation, using models of acute and chronic segmental ischemic LV dysfunction evaluated by three-dimensional (3D) echocardiography. BACKGROUND: Dysfunction and dilation-both mechanisms with practical therapeutic implications-are difficult to separate in patients. METHODS: In seven dogs with acute left circumflex (LCX) coronary ligation, LV expansion was initially restricted and then permitted to occur. In seven sheep with LCX branch ligation, LV expansion was also initially limited but became prominent with remodeling over eight weeks. Three-dimensional echo reconstruction quantified mitral apparatus geometry and MR volume. RESULTS: In the acute model, despite LV dysfunction with ejection fraction = 23 +/- 8%, MR was initially trace with limited LV dilation, but it became moderate with subsequent prominent dilation. In the chronic model, MR was also initially trace, but it became moderate over eight weeks as the LV dilated and changed shape. In both models, the only independent predictor of MR volume was increased tethering distance from the papillary muscles (PMs) to the anterior annulus, especially medial and posterior shift of the ischemic medial PM, measured by 3D reconstruction (r2 = 0.75 and 0.86, respectively). Mitral regurgitation volume did not correlate with LV ejection fraction or dP/dt. CONCLUSIONS: Segmental ischemic LV contractile dysfunction without dilation, even in the PM territory, fails to produce important MR. The development of MR relates strongly to changes in the 3D geometry of the mitral apparatus, with implications for approaches to restore a more favorable configuration.

Inter- and intra-specific gene-density-correlated radial chromosome territory arrangements are conserved in Old World monkeys.

Recently it has been shown that the gene-density correlated radial distribution of human 18 and 19 homologous chromosome territories (CTs) is conserved in higher primates in spite of chromosomal rearrangements that occurred during evolution. However, these observations were limited to apes and New World monkey species. In order to provide further evidence for the evolutionary conservation of gene-density-correlated CT arrangements, we extended our previous study to Old World monkeys. They comprise the remaining species group to be analyzed in order to obtain a comprehensive overview of the nuclear topology of human 18 and 19 homologous CTs in higher primates. In the present study we investigated four lymphoblastoid cell lines from three species of Old World monkeys by three-dimensional fluorescence in situ hybridization (3D-FISH): two individuals of Japanese macaque (Macaca fuscata), crab-eating macaque (Macaca fascicularis), and an interspecies hybrid individual between African green monkey (Cercopithecus aethiops) and Patas monkey (Erythrocebus patas). Our data demonstrate that gene-poor human 18 homologous CTs are located preferentially close to the nuclear periphery, whereas gene-dense human 19 homologous CTs are oriented towards the nuclear center in all cell lines analyzed. The gene-density-correlated positioning of human 18 and 19 homologous CTs is evolutionarily conserved throughout all major higher primate lineages, despite chromosomal inversions, fusions, fissions or reciprocal translocations that occurred in the course of evolution in these species. This remarkable preservation of a gene-density-correlated chromatin arrangement gives further support for a functionally relevant higher-order chromatin architecture.

Identification of a gene that shortens clonal life span of Paramecium tetraurelia.

We have isolated a Paramecium tetraurelia mutant that divides slowly in daily reisolation cultures and repeats short clonal life spans after successive autogamies. Here we show, using breeding analysis, that a recessive mutation is responsible for the low fission rate and that this low rate is closely related to the short clonal life span. We conclude that a single pleiotropic gene controls these traits and have named it jumyo. In an attempt to further characterize the jumyo mutant, we have revealed that it has a culture life span similar to that of the wild-type cells and that, when mass cultured, it can divide as rapidly as wild-type cells. There was strong evidence that the mutant cells excreted into culture medium some substance that promotes their cell division. These findings may not only present supporting evidence for the hypothesis that the cellular life span is genetically programmed but also give a material basis for the study of the controlling mechanism of cell division in relation to the clonal life span.