A new application of the phase-field method for understanding the mechanisms of nuclear architecture reorganization.

Specific features of nuclear architecture are important for the functional organization of the nucleus, and chromatin consists of two forms, heterochromatin and euchromatin. Conventional nuclear architecture is observed when heterochromatin is enriched at nuclear periphery, and it represents the primary structure in the majority of eukaryotic cells, including the rod cells of diurnal mammals. In contrast to this, inverted nuclear architecture is observed when the heterochromatin is distributed at the center of the nucleus, which occurs in the rod cells of nocturnal mammals. The inverted architecture found in the rod cells of the adult mouse is formed through the reorganization of conventional architecture during terminal differentiation. Although a previous experimental approach has demonstrated the relationship between these two nuclear architecture types at the molecular level, the mechanisms underlying long-range reorganization processes remain unknown. The details of nuclear structures and their spatial and temporal dynamics remain to be elucidated. Therefore, a comprehensive approach, using mathematical modeling, is required, in order to address these questions. Here, we propose a new mathematical approach to the understanding of nuclear architecture dynamics using the phase-field method. We successfully recreated the process of nuclear architecture reorganization, and showed that it is robustly induced by physical features, independent of a specific genotype. Our study demonstrates the potential of phase-field method application in the life science fields.

Hypoxic hepatitis with marked elevation of serum ferritin probably due to activation of intrahepatic macrophages: another form of hypoxic hepatitis hitherto not reported?

BACKGROUND AND STUDY AIMS: Hypoxic hepatitis (HH) is an acute liver injury that develops in patients with underlying diseases, such as heart failure, respiratory failure, septic/toxic shock. However, some patients do not have underlying diseases or episodes which are known to result in HH. Here, we analyzed the clinical characteristics of this particular patient group (called 'unknown HH' hereafter) to understand its pathogenesis. PATIENTS AND METHODS: Between October 2010 and January 2016, 157 consecutive patients with acute liver injury were admitted to our hospital. Among these patients, 15 patients were categorized as unknown HH. Medical histories and blood test results of unknown HH were analyzed. RESULTS: Among 15 patients of unknown HH, 11 were habitual drinkers and all experienced one of digestive symptoms which might result in mild hypovolemia such as vomiting, diarrhea, appetite loss, and epigastralgia. All patients of unknown HH presented marked elevation of serum ferritin concentration paralleled with aspartate transaminase (AST), alanine transaminase (ALT), and lactate dehydrogenase (LDH) concentrations. The serum levels of ferritin, ALT, LDH, and prothrombin time-international normalized ratio (PT-INR) were rapidly decreased during hospitalization and all 15 patients of unknown HH recovered without any complication. CONCLUSIONS: We found the particular group of HH with marked elevation of serum ferritin probably due to intrahepatic macrophage activation. Anti-inflammatory treatments might be effective for this group of hypoxic hepatitis.

Rad51 accumulation at sites of DNA damage and in postreplicative chromatin.

Rad51, a eukaryotic RecA homologue, plays a central role in homologous recombinational repair of DNA double-strand breaks (DSBs) in yeast and is conserved from yeast to human. Rad51 shows punctuate nuclear localization in human cells, called Rad51 foci, typically during the S phase (Tashiro, S., N. Kotomura, A. Shinohara, K. Tanaka, K. Ueda, and N. Kamada. 1996. Oncogene. 12:2165-2170). However, the topological relationships that exist in human S phase nuclei between Rad51 foci and damaged chromatin have not been studied thus far. Here, we report on ultraviolet microirradiation experiments of small nuclear areas and on whole cell ultraviolet C (UVC) irradiation experiments performed with a human fibroblast cell line. Before UV irradiation, nuclear DNA was sensitized by the incorporation of halogenated thymidine analogues. These experiments demonstrate the redistribution of Rad51 to the selectively damaged, labeled chromatin. Rad51 recruitment takes place from Rad51 foci scattered throughout the nucleus of nonirradiated cells in S phase. We also demonstrate the preferential association of Rad51 foci with postreplicative chromatin in contrast to replicating chromatin using a double labeling procedure with halogenated thymidine analogues. This finding supports a role of Rad51 in recombinational repair processes of DNA damage present in postreplicative chromatin.

L-leucine: a neuroactive substance in insects.

A compound was isolated from the blood of silkworm larvae, Bombyx mori, which had been prostrated with DDT; this compound increased the spontaneous discharge in the isolated abdominal nerve cord of the American cockroach, Periplaneta americana. The compound was identified as L-leucine.

Molecular cloning reveals that the p160 Myb-binding protein is a novel, predominantly nucleolar protein which may play a role in transactivation by Myb.

We have previously detected two related murine nuclear proteins, p160 and p67, that can bind to the leucine zipper motif within the negative regulatory domain of the Myb transcription factor. We now describe the molecular cloning of cDNA corresponding to murine p160. The P160 gene is located on mouse chromosome 11, and related sequences are found on chromosomes 1 and 12. The predicted p160 protein is novel, and in agreement with previous studies, we find that the corresponding 4.5-kb mRNA is ubiquitously expressed. We showed that p67 is an N-terminal fragment of p160 which is generated by proteolytic cleavage in certain cell types. The protein encoded by the cloned p160 cDNA and an engineered protein (p67*) comprising the amino-terminal region of p160 exhibit binding specificities for the Myb and Jun leucine zipper regions identical to those of endogenous p160 and p67, respectively. This implies that the Myb-binding site of p160 lies within the N-terminal 580 residues and that the Jun-binding site is C-terminal to this position. Moreover, we show that p67* but not p160 can inhibit transactivation by Myb. Unexpectedly, immunofluorescence studies show that p160 is localized predominantly in the nucleolus. The implications of these results for possible functions of p160 are discussed.

Anticancer effects of local administration of mitomycin C via the hepatic artery or portal vein on implantation and growth of VX2 cancer injected into rabbit liver.

Rabbits were inoculated with a suspension of VX2 carcinoma cells in the liver, and mitomycin C was given via the hepatic artery or the portal vein for a study of the anticancer effects. Twenty-eight rabbits were killed for preliminary study at 1 h or 1, 3, 7, 9, 12, or 14 days after the inoculation. Another 36 rabbits were divided into three groups. Groups A and B were given the agent (0.5 mg/kg), 1 h after the inoculation and on Days 2, 4, 6, and 8, into the common hepatic artery or the splenic vein, respectively. Group C was not treated after inoculation. The mean numbers of cancer nodules per rabbit in Groups A, B, and C were 11.9, 36.4, and 83.4, respectively, at 12 days after inoculation. The number of cancer nodules of Group A was smallest (P less than 0.025, F test). The means of the total cross-sectional area of tumor nodules in Groups A, B, and C were 32.7, 79.7, and 217.3 mm2, respectively. The total cross-sectional area of the cancer nodules of Group A was smallest (P less than 0.05, F test). These results suggest that the anticancer agents given via the hepatic artery had better effects on early (small) metastatic liver tumor than those via the portal vein.

Cellular retinoic acid-binding protein in virus-induced Shope papillomas of rabbit skin.

Cellular retinoic acid-binding protein (cRABP) was detected in the cytosol of virus-induced papilloma (Shope) of rabbit skin. The Shope papilloma cRABP showed the same ligand specificity and sedimentation value (2S) as was found in other animal species. The level of cRABP in the papillomatous tissue was significantly higher than that in the normal rabbit skin and increased in accordance with the growth and development of the tumor, reaching a peak about 40 days after the inoculation of the Shope papilloma virus. Although this binding capacity was about 15 times greater than in the normal skin, the level of cRABP in the transplantable carcinomas Vx2 and Vx7, both originating from the virus-induced papillomas over 20 years ago, was much the same as in normal rabbit skin.

Analysis of individual differences in radiosensitivity using genome editing.

Current standards for radiological protection of the public have been uniformly established. However, individual differences in radiosensitivity are suggested to exist in human populations, which could be caused by nucleotide variants of DNA repair genes. In order to verify if such genetic variants are responsible for individual differences in radiosensitivity, they could be introduced into cultured human cells for evaluation. This strategy would make it possible to analyse the effect of candidate nucleotide variants on individual radiosensitivity, independent of the diverse genetic background. However, efficient gene targeting in cultured human cells is difficult due to the low frequency of homologous recombination (HR) repair. The development of artificial nucleases has enabled efficient HR-mediated genome editing to be performed in cultured human cells. A novel genome editing strategy, 'transcription activator-like effector nuclease (TALEN)-mediated two-step single base pair editing', has been developed, and this was used to introduce a nucleotide variant associated with a chromosomal instability syndrome bi-allelically into cultured human cells to demonstrate that it is the causative mutation. It is proposed that this editing technique will be useful to investigate individual radiosensitivity.

Cell type-specific trans-activation by the B-myb gene product: requirement of the putative cofactor binding to the C-terminal conserved domain.

The myb gene family has three members, c-myb, A-myb and B-myb. We have examined the trans-activating capacity of the B-myb gene product (B-Myb) in various types of cells. B-Myb functions as a transcriptional activator in CV-1 and HeLa cells, but not in NIH3T3 cells, indicating that B-Myb is a cell type-specific transcriptional activator. Deletion analyses of B-Myb have demonstrated that the region conserved between three members of the myb gene family (CR for conserved region) is necessary for trans-activation by B-Myb. An in vivo competition assay suggests that regulatory factor(s) that binds to the CR of B-Myb is required for transactivation. Analyses using an affinity resin show that multiple proteins bind to the CR of B-Myb and that the CR-binding proteins in CV-1 and HeLa cells are different from those in NIH3T3 cells. These results suggest that the CR-binding cofactor(s) is critical for the cell type-specific trans-activation by B-Myb.

S phase specific formation of the human Rad51 protein nuclear foci in lymphocytes.

The Rad51 protein, which is a homologue of the bacterial RecA protein, is involved in mitotic and meiotic recombination and in repair of double-strand breaks of DNA in yeast. The Rad51 homologue is conserved from yeast to human. In this study, the Rad51 protein was shown to be induced in peripheral blood lymphocytes (PBLs) 36 h after phytohemagglutinin (PHA) stimulation. Immunofluorescence study revealed that the distribution of the Rad51 protein in the nucleus was not uniform and focus-like staining was observed. Formation of the Rad51 foci was induced at 36 h after treatment of the cells with PHA. Twenty five percent of the cells had the foci at this time and the number of cells with foci declined thereafter. Cell cycle study using laser microscope by double staining method suggested that the appearance of the Rad51 nuclear foci was S phase specific. Furthermore, double staining study for the Rad51 protein and incorporated BrdU confirmed S phase specific appearance of the Rad51 nuclear foci. Formation of the Rad51 nuclear foci in PHA-stimulated lymphocytes might be involved in DNA recombination or DNA repair in S phase. The roles of RAD51 foci in S-phase will be discussed.

Identification of illegitimate recombination hot spot of the retinoic acid receptor alpha gene involved in 15;17 chromosomal translocation of acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) has been characterized by 15;17 chromosomal translocation, which involves the retinoic acid receptor alpha (RARA) gene on chromosome 17 and the PML gene on chromosome 15. The breakpoints have been mapped to three cluster regions in the PML gene, and to RARA gene intron 2. We have examined the distribution of breakpoints within RARA gene intron 2. An extremely restricted region (ERR) of 50 bp within RARA gene intron 2 was identified as the cluster region of breakpoints by polymerase chain reaction and sequence analysis of DNA from APL patients. To study experimentally the mechanism involved in the translocation, ERR was tested in NIH3T3 cells by in vitro transfection-recombination assay, in which target sequences were placed either downstream of the SV40 promoter or upstream of the neo gene. Cells were conferred resistance to G418 only when the promoter was fused to the neo gene by recombination of two target sequences during transfection. The molecular junctions were analysed in five clones, and all of them were shown to be confined within a 20 bp region in a 148 bp DNA fragment containing ERR. This suggests that ERR might be the illegitimate recombination hot spot in mammalian cells.

Cytogenetic 2; 18 and 18; 22 translocation in chronic lymphocytic leukemia with juxtaposition of bcl-2 and immunoglobulin light chain genes.

The majority of follicular lymphoma cells carry the typical chromosome translocation 14;18, which juxtaposes the bcl-2 gene to the immunoglobulin heavy-chain (IgH) gene. Variant translocations of the bcl-2 gene to the Ig lambda or Ig kappa gene have been found by molecular biological techniques in a significant fraction (approximately 10%) of chronic lymphocytic leukemia (CLL). However, there have been no reports describing the presence of cytogenetic 18;22 and 2;18 translocations in CLL, in spite of extensive karyotypic studies. We present here two cases of CLL, one with cytogenetically detected t(2;18)(p11;q21) and the other with the t(18;22)(q21;q11). The molecular analysis revealed that these translocations juxtaposed the bcl-2 and immunoglobulin light-chain (IgL) genes. The t(18;22) broke the 5' flanking region of the bcl-2 gene and juxtaposed to the immunoglobulin lambda light-chain (Ig lambda) gene in a head-to-head configuration, as in the cases previously described. In the case of the t(2;18), the bcl-2 gene and immunoglobulin kappa light-chain (Ig kappa) gene were juxtaposed in a head-to-tail configuration, which is opposite to that expected from the orientation of the genes on chromosomes. The breakpoint was located within the 5' untranslated region of the bcl-2 gene. The results presented here indicate that the bcl-2/immunoglobulin light-chain (IgL) gene juxtaposition seen in a fraction of CLL is the result of cytogenetically detectable reciprocal chromosome translocations 2;18 and 18;22.

Clinical features of isolated noncompaction of the ventricular myocardium: long-term clinical course, hemodynamic properties, and genetic background.

OBJECTIVES: A nationwide survey was conducted to clarify the clinical features of isolated noncompaction of the ventricular myocardium (INVM) in Japanese children in comparison with features previously described in patients with INVM. BACKGROUND: Isolated noncompaction of the ventricular myocardium is a rare disorder characterized by an excessively prominent trabecular meshwork. It is accompanied by depressed ventricular function, systemic embolism and ventricular arrhythmia. METHODS: A questionnaire specifically designed for this study was sent to 150 hospitals in Japan where a pediatric cardiology division exists. RESULTS: Twenty-seven patients were diagnosed by two-dimensional echocardiography, their ages ranging from one week to 15 years at presentation, with follow-up lasting as long as 17 years. The gross anatomical appearance and the extension of noncompacted myocardium predominantly at the apex observed on two-dimensional echocardiograms were similar to observations reported previously. Dissimilarities included a greater number of asymptomatic patients at initial presentation, a longer clinical course with gradually depressed left ventricular function, no systemic embolism, and rare ventricular tachycardia in the Japanese children. Cardiac catheterization disclosed normal left ventricular end-diastolic volume and increased left ventricular end-diastolic pressure in most cases, consistent with restrictive hemodynamics. A higher incidence of Wolff-Parkinson-White syndrome was found in the children, whereas left bundle branch block was rarer than reported in adults. Familial recurrence was high (44%) and included many women. CONCLUSIONS: In Japanese children, INVM can be found by screening examinations at asymptomatic stage, and it might have a longer dinical course with gradually depressed left ventricular function and restrictive hemodynamics. The pattern of familial recurrence we observed implies that INVM is a distinctive clinical entity with a heterogeneous genetic background.

Nuclear proteins binding to the recombination hotspot region of the retinoic acid receptor alpha gene.

Acute promyelocytic leukemia (APL) has been characterized by 15;17 chromosomal translocation, which involves the retinoic acid receptor alpha (RARA) gene on chromosome 17 and the PML gene on chromosome 15. An extremely restricted region (ERR) of 50 bps within the second intron of the RARA gene was identified as the cluster region of breakpoints by sequencing analyses. ERR was tested by in vitro transfection-recombination assay, and was shown to be the recombination hot spot. In this study, presence of DNA binding proteins to the 148 bps DNA fragment which contains ERR was confirmed by gel-mobility shift analysis in the nuclear extract of NIH3T3 cells and human leukemia cell lines. Furthermore, in vitro study with the mouse sarcoma cell lines using the recombination reporter plasmid containing ERR showed that ERR might be involved in the homologous recombination in addition to the illegitimate recombination. The DNA binding proteins specific to ERR might play an important role in chromosome translocation.

Assessment of the biological malignancy of hepatocellular carcinoma: relationship to clinicopathological factors and prognosis.

It is difficult to determine the prognosis of patients with hepatocellular carcinoma (HCC). Assessment of the clinicopathological and biological malignancy of HCC may help in determining treatment strategies and predicting outcome. The tumor DNA content, p53 protein expression, proliferating cell nuclear antigen labeling index, and argyrophilic proteins of nuclear organizer regions were used as markers of biological malignancy. A correlation between these biological parameters and clinicopathological factors was sought. DNA aneuploidy was observed in 31 of 80 tumors (38.8%). Aneuploidy increased as differentiation decreased. The overall survival rate of patients with aneuploid tumors was significantly poorer than that of patients with diploid tumors. p53 overexpression was observed in 18 of 80 tumors (22.5%). The incidence of p53 positivity increased significantly with increasing tumor size and poorer differentiation. The overall survival rate of p53-positive patients was significantly worse than that of p53-negative patients. The proliferating cell nuclear antigen labeling index and the mean number of argyrophilic proteins of nuclear organizer regions were higher in more poorly differentiated lesions. We conclude that DNA ploidy and p53 expression are useful prognostic indicators in HCC. Cell proliferation increases as HCC progresses. With progression, tumors tend to become more poorly differentiated.

Structure and expression of hedgehog, a Drosophila segment-polarity gene required for cell-cell communication.

The complete nucleotide sequence of the coding region of hedgehog (hh), a segment-polarity gene in Drosophila melanogaster, was determined. The gene was found to include three exons which would encode a 421- (or 471-) amino acid (aa) polypeptide with a long hydrophobic stretch. The hh mRNA was about 2.3 kb long and expressed throughout development. The hh expression in an embryo occurred in stripes, while that in imaginal discs occurred in the posterior compartment. As a whole, the spatial expression pattern of hh mRNA was very similar to that of engrailed (en), a homeobox gene required for the formation of the anterior-posterior compartment boundary. Unlike en, no hh expression was observed in the central nervous system.

Multiple nuclear localization signals of the B-myb gene product.

Nuclear entry of the B-myb gene product (B-Myb) is dependent on multiple nuclear localization signals (NLS's). Mutagenesis of the putative NLS's of B-Myb has identified two separate NLS's, NLS1 and NLS2. Each of the two NLS's is essential for efficient nuclear targeting. NLS2 contains two interdependent basic domains separated by 8 intervening spacer amino acids, and both basic domains are required for nuclear entry. Thus, NLS2 belongs to a class of bipartite NLS's. Like the NLS's in yeast transcription factor SW15, NLS2 contains a putative cdc2 kinase site. However, unlike the case of SW15, phosphorylation at this site did not affect the nuclear targeting of B-Myb.

The effect of perioperative portal venous injection of donor spleen cells on renal allograft survival in dogs.

Preoperative portal venous injection of donor lymphocytes has been shown to extend allograft survival in inbred animals. However, the effect of perioperative injection in a large animal model has not been carefully studied. We examined the effect of perioperative (day 0) donor spleen cell administration in a canine kidney transplant model using both a double-donor transplantation and a single-donor survival study. In the double-donor model, two kidneys from two separate donors were transplanted into a single recipient. Spleen cells obtained from only one of the donors were injected through the portal vein immediately after reperfusion of the allografts. Both allografted kidneys were resected 14 days after transplantation and studied. There was only a slight cellular infiltrate in the kidney derived from the donor from whom spleen cells had been obtained, whereas a much more extensive cellular infiltrate as well as edema and hemorrhage were present in the other kidney. In a survival study of single allografts, the injection of 2 x 10(9) spleen cells significantly prolonged the mean survival time (15.0 +/- 3.4 days) compared with untreated recipients (6.6 +/- 2.2 days). Addition of cyclosporine did not significantly prolong survival in the spleen cell inoculated animals. These findings suggest that perioperative protal venous injection of donor cells may be a specific immunosuppressive method useful in clinical organ transplantation.

Effect of donor-specific splenocytes via portal vein and FK506 in rat small bowel transplantation.

BACKGROUND: To investigate the role of the liver in immune responses after small bowel transplantation, donor-specific splenocytes were infused perioperatively, via the portal vein, in a rat heterotopic small bowel transplant model. METHODS: Heterotopic small bowel transplantation between the fully allogenic Brown Norway (BN) (RT1n) and Lewis (RT1[1]) strain rats were performed. We prepared donor splenocytes from BN or third-party WKA (RT1k) rat spleens for Lewis hosts and injected the splenocytes perioperatively via the host portal vein or the systemic vein. The hosts were treated with a short course of the immunosuppressive agent, FK506 (0.5 mg/kg, 0-3 days postoperatively), following the experimental protocols. RESULTS: Untreated Lewis hosts rejected BN small bowel grafts at 5.4+/-0.9 days (n=8). BN splenocytes given alone caused fatal graft-versus-host disease in six of eight animals, and two others died from graft rejection. FK506 alone did not significantly prolong graft survival (6.3+/-1.0 days, n=10). However, BN splenocytes injected via the portal vein, combined with FK506, prolonged graft survival to 12.7+/-2.1 days (n=12, P < 0.01) and 10 of 12 rats survived more than 70 days. This was donor antigen specific. BN splenocytes administered systemically caused fatal graft-versus-host disease in all recipients, and FK506 did not ameliorate this. Histologic findings of graft rejection were remarkably mild in the recipients of the combined therapy, compared with the recipients that were given FK506 alone. Down-regulation of one-way mixed lymphocyte reaction to BN splenocytes was observed in the splenocytes of the tolerant hosts. CONCLUSIONS: Combined administration of donor splenocytes and FK506 reduced allograft rejection and prolonged survival in this rat model of small bowel transplantation.

Acetaminophen absorption test as a marker of small bowel transplant rejection.

We recently evaluated the acetaminophen absorption test as a marker of graft rejection for small bowel transplantation(SBTX). Randomly bred male Wistar rats were used as recipients and donors. Rats (n=45) received heterotopic small intestinal transplants and were divided into three groups (n=15 for each group). In group A, a 10-cm segment of jejunum of was exteriorized as a Thiry-Vella loop. In group B, immunosuppression was not given after SBTX. In group C, rats were treated with FK506 after SBTX (0.3 mg/kg body weight, 0-6 postoperative days). Serum acetaminophen concentrations were measured 15 min after instillation of 0.15 g/kg acetaminophen into the intestinal loop on postoperative days 1, 3, and 7 (n=5 for each group). Blood flow and histology of the graft were also evaluated. In the SBTX group only, the grafts showed the histological change after acute rejection. On day 3, plasma acetaminophen concentrations in this group showed a significant decrease, which correlated with the mild histological changes of graft rejection. Graft blood flow of the SBTX group decreased significantly on day 7, following the severe graft destruction of advanced rejection. No remarkable changes were observed in the other two groups. The acetaminophen absorption test appears to be useful for the early detection of SBTX graft rejection.