Tenascin-C expression contributes to pediatric brainstem glioma tumor phenotype and represents a novel biomarker of disease.

Diffuse intrinsic pontine glioma (DIPG), an infiltrative, high grade glioma (HGG) affecting young children, has the highest mortality rate of all pediatric cancers. Despite treatment, average survival is less than twelve months, and five-year survival under 5%. We previously detected increased expression of Tenascin-C (TNC) protein in DIPG cerebrospinal fluid and tumor tissue relative to normal specimens. TNC is an extracellular matrix (ECM) glycoprotein that mediates cell-matrix interactions, guides migrating neurons during normal brain development and is thought to maintain the periventricular stem cell niche in the developing brain. Tumor TNC expression is reported in adult glioma and other cancers. However, the pattern and effects of TNC expression in DIPG has not been previously explored. Here, we characterize TNC expression in patient derived pediatric supratentorial HGG (n = 3) and DIPG (n = 6) cell lines, as well as pediatric glioma tumor (n = 50) and normal brain tissue specimens (n = 3). We found tumor specific TNC gene and protein overexpression that directly correlated with higher tumor grade (WHO III and IV, p = 0.05), H3K27 M mutation (p = 0.012), shorter progression free survival (p = 0.034), and poorer overall survival (0.041) in association with these factors. TNC knockdown via lentiviral shRNA transfection of HGG (n = 1) and DIPG (n = 3) cell lines resulted in decreased cell proliferation, migration, and invasion in vitro (p < 0.01), while TNC cDNA transfection resulted in increased cell migration, invasion and proliferation (p < 0.01) as well as altered cell morphology in H3K27 M mutant DIPG lines. Whole transcriptome sequencing analysis (RNA-Seq) on DIPG (n = 3) and HGG (n = 2) cell lines after TNC cDNA, shRNA, and empty vector control transfection revealed the effects of TNC expression level on global gene expression profiles. Together, our findings reveal TNC expression in DIPG in association with H3K27 M mutation and VEGF signaling, and suggest that TNC may contribute to DIPG tumor phenotype, and serve as a clinically detectable biomarker for DIPG.

A doxycycline loaded, controlled-release, biodegradable fiber for the treatment of aortic aneurysms.

The pathogenesis of aortic aneurysm (AA) is characterized by degradation of extracellular matrix with increased matrix metalloproteinases (MMPs) and inflammatory reaction. Doxycycline (DOXY) has been reported to control the extension of AA by regulation of MMP. However, systemic administration may cause adverse side effects. In this study, we demonstrated the possibility of local administration of DOXY controlled-release biodegradable fiber (DCRBF) for AA in mice. DCRBF was fabricated by biodegradable polymer (polylactic acid; PLA) mixed with DOXY using an electrospinning technique. DCRBF was cocultured with SMCs, macrophages and aortic tissue, and placed on an abdominal aortic aneurysm which induced apolipoprotein E-deficient mice. We evaluated gene and protein expression of proteases, elastin and inflammatory markers. In the presence of DCRBF, MMP-12 was significantly decreased, TGF-ß1 and Lox were significantly increased in SMC gene expression, MMP-9 and -12 significantly decreased gene expression of macrophages. The DCRBF preserved elastin content and decreased MMP-2 and -9 in aortic tissue. In addition, IGF-1 and TIMP-1 were significantly increased and IL-6 and TNF-α were significantly decreased with DCRBF in vivo. In conclusion, our results suggested that local administration of DCRBF may become a promising alternative therapeutic strategy for AA.

[Usefulness of semi-skeletonized right gastroepiploic artery].

The right gastroepiploic artery (RGEA) could be harvested easily and safely by using an ultrasonic scalpel. It is easier and faster not to remove a satellite vein from RGEA than to skeletonize it fully. And blood flow of the vein is important, because it is the flow of vasa vasorum of RGEA. Among 70 patients who have bypassed to right coronary arteries (RCA), 25 patients were treated with semi-skeletonized RGEA (SSK-RGEA), the other 45 patients were operated with pedicled RGEA or (if pedicled RGEA was short and small) with the other grafts. An availability of SSK-RGEA for RCA was 100%. And that of pedicled RGEA was 47% (p < 0.001). Twenty-eight patients were operated without a pump. Twenty-three of them (82%) were bypassed with RGEA. In the on-pump cases (42 patients), RGEA were used for 13 (31%) cases (p < 0.05). Early post-operative angiographies revealed 1 occlusion. But the site of occlusion was the origin of RGEA branch from a gastro-duodenal artery, and the anastomotic site was patent. This graft was supposed to be occluded post-operatively by arteriosclerosis. Flow competition occurred in two grafts. In conclusion, the SSK-RGEA was useful for RCA bypass grafting. The reliability of RGEA should increase the indication of off-pump coronary artery bypass grafting.

[Pitfall in the choice of the in situ arterial grafts in OPCAB].

Flow capacity with arterial grafts which have been used in off-pump CABG has been discussed. To analyze the hypoperfusion syndrome when in situ arterial grafts are selected, we made the simple mathematical model which consisted of two vessels with in parallel. We speculated the flow capacity of the various grafts, and flow distribution in the distal coronary artery, based on Poiseuille's law. This theoretical model clearly demonstrated that hypoperfusion syndrome occurred in reoperative coronary bypass grafting had a close relation with the diameters and lengths of bypass grafts.

The RING heterodimer BRCA1-BARD1 is a ubiquitin ligase inactivated by a breast cancer-derived mutation.

BRCA1-BARD1 constitutes a heterodimeric RING finger complex associated through its N-terminal regions. Here we demonstrate that the BRCA1-BARD1 heterodimeric RING finger complex contains significant ubiquitin ligase activity that can be disrupted by a breast cancer-derived RING finger mutation in BRCA1. Whereas individually BRCA1 and BARD1 have very low ubiquitin ligase activities in vitro, BRCA1 combined with BARD1 exhibits dramatically higher activity. Bacterially purified RING finger domains comprising residues 1-304 of BRCA1 and residues 25-189 of BARD1 are capable of polymerizing ubiquitin. The steady-state level of transfected BRCA1 in vivo was increased by co-transfection of BARD1, and reciprocally that of transfected BARD1 was increased by BRCA1 in a dose-dependent manner. The breast cancer-derived BARD1-interaction-deficient mutant, BRCA1(C61G), does not exhibit ubiquitin ligase activity in vitro. These results suggest that the BRCA1-BARD1 complex contains a ubiquitin ligase activity that is important in prevention of breast and ovarian cancer development.

C-erbB-2/ HER-2 upregulates fascin, an actin-bundling protein associated with cell motility, in human breast cancer cell lines.

The over-expression of c-erbB-2/ HER-2, a receptor tyrosine kinase, correlates with poor prognosis in patients with breast and ovarian cancer. In the human breast cancer cell line, MDA-MB-435, c-erbB-2 over-expression results in increased chemoinvasion and higher metastatic properties in nude mice. However, the mechanisms by which c-erbB-2 increases the malignant potential of cells remains unclear. We have determined that over-expression of c-erbB-2 in MDA-MB-435 cells, and in some additional breast cancer cell lines, is associated with graphic increases in mRNA and protein levels of the actin bundling protein fascin. Heightened fascin expression has been observed in other systems to result in greatly increased cell motility, and indeed, our work employing semi-automated time-lapse microscopy demonstrates that MDA-MB-435 cells over-expressing c-erbB-2 exhibit significantly heightened cellular dynamics and locomotion, while visualization of bundled microfilaments within fixed cells revealed enhanced formation of dendritic-like processes, microspikes and other dynamic actin based structures. To address the means by which c-erbB-2 over-expression might result in elevated fascin levels, we identified multiple perfect match TCF and NF-kappaB consensus sites in fascin's promoter and first intron, which appeared consistent with the greater endogenous transcriptional activities of TCF and NF-kappaB in c-erbB-2 over-expressing MDA-MB-435 cells. While such transcriptional modulation may occur in the context of the intact gene/chromatin, subsequent tests using reporter constructs did not support involvement of these signaling pathways. In conclusion, highly increased fascin levels were observed in MDA-MB-435 over-expressing c-erbB-2, likely contributing to these cells' altered actin dynamics, and increased cell motility and malignancy. Studies in progress aim to discern the means by which c-erbB-2 over-expression leads to transcriptional activation of the fascin gene.

Fascin, an actin-bundling protein associated with cell motility, is upregulated in hormone receptor negative breast cancer.

Loss of hormone receptor (HR) status in breast carcinomas is associated with increased tumour cell motility and invasiveness. In an immunohistological study of 58 primary breast cancers, oestrogen (ER) and progesterone (PR) receptor levels were inversely correlated with the expression of fascin, an actin-bundling protein associated with cell motility (P< 0.0001 and P = 0.0019, respectively). In addition, fascin was preferentially expressed in non-diploid tumours (P = 0.03). In summary, the upregulation of fascin in HR-negative breast cancers may contribute to their more aggressive behaviour.

NER rat strain: a new type of genetic model in epilepsy research.

PURPOSE: We characterized and evaluated as an animal model of epilepsy NER, a new epileptic rat strain, which was developed by inbreeding rats with spontaneous tonic-clonic seizures in a stock of Crj:Wistar. METHODS: Animals were monitored through the inbreeding course, and video-EEGs were recorded selectively. External seizure-provoking stimuli were applied to NER and to a control parental strain. F1, F2, and backcross progenies were produced between NER and a nonepileptic unrelated strain. Pathologic study included hematoxylin-and-eosin (HE), Klüver-Barrera's, modified Bodian silver, and neo-Timm's staining. RESULTS: After the F9 generation, 94%-98% of NER exhibited spontaneous tonic-clonic convulsions, beginning with neck and forelimb clonus, wild jumping/running, opisthotonic posturing, and evolving to tonic, then clonic convulsion, followed by postictal flaccidity. Most seizure onsets occurred between 2-4 months of age, and the incidence was 0.45 +/- 0.21 seizures in 12 h. Ictal cortical and hippocampal EEGs were characterized by high-voltage spikes followed by diffuse spike-and-wave or polyspike-and-wave complexes. NER revealed seizure susceptibility to pentylenetetrazol, tossing, and transcorneal electroshock, but not to tactile, photic, or acoustic stimuli, or to transauricular electroshock. Mating experiments revealed that 0% (0/46) of the animals in F1, 25.5% (13/51) in F2, and 63.6% (56/88) in backcross progenies exhibited spontaneous tonic-clonic convulsions without sex difference. For all these epileptic traits, no pathologic changes were demonstrated in the CNS. CONCLUSIONS: NER frequently exhibited spontaneous convulsions, controlled by a major autosomal recessive gene for epilepsy, that are comparable to generalized tonic-clonic seizures in humans. This can serve as a new genetic model in epilepsy research.

[Continuous hepatic arterial infusion of fluorouracil with leucovorin for liver metastases from colorectal cancer].

Sixteen patients with liver metastases from colorectal cancer were treated by continuous intraarterial chemotherapy of 5-FU and leucovorin. The regimen was that 500 mg/body of 5-FU with 30 mg/ body of leucovorin was administered continuously for 5 days followed by no medication for 16 days. The effect of this therapy was evaluated, and the relationship between this therapy and p53 overexpression was also studied. A complete response was obtained in 3 patients and a partial response in 3 patients; the overall response rate was 38%. The response rate was 56% in patients of more than 6 courses, 57% in patients with positive p53, and 20% in patients with negative p53. The three-year survival rate was 31%, and median survival was 18 months. Duodenal ulcer occurred in 2 patients due to extravascular dislocation of catheter. A high response rate and favorable prognosis were obtained by this therapy. Maintenance of catheter and a short administration period are current issues.

Analysis of Cdc2 and Cyclin D1 Expression in Breast Cancer by Immunoblotting.

Cyclins and cyclin-dependent kinases may reflect the status of cell proliferation in cancer tissues. The authors sought to determine whether cdc2 and cyclin D1 are expressed in breast cancer and are useful as prognostic factors. Accumulation of cdc2 and cyclin D1 proteins was examined in 88 cases of breast cancer using immunoblotting techniques and correlations with clinicopathological factors and prognoses were investigated. Cdc2 and cyclin D1 proteins were observed in 27.3 % and 75.0 % of breast cancers studied, respectively. The incidence of lymph node metastasis was significantly high in cdc2/cyclin D1-double positive group and low in double negative group. On the other hand, the incidence of estrogen receptor (ER) negative cases was significantly higher in the cdc2-positive/cyclin D1-negative group. Relapse-free survival times of cdc2-positive cases were significantly shorter than those of Cdc2-negative cases. The relapse-free survival times of cyclin D1-positive cases also tended to be poorer than those of cyclin D1-negative cases. Multivariate analyses revealed cdc2 as the second most significant of the prognostic variables, following lymph node status. The three-year relapse-free survival rate of cdc2/cyclin D1-double positive cases was 58.9%, whereas that of cdc2/cyclin D1-doublue negative cases was 100%. Cdc2 and cyclin D1 represent the status of cell proliferation in breast cancer, and may be useful in breast cancer assessment.

Expression of beta-catenin in normal breast tissue and breast carcinoma: a comparative study with epithelial cadherin and alpha-catenin.

Expression of beta-catenin was investigated in normal breast tissue and 66 breast carcinomas in conjunction with expression of epithelial cadherin (E-CD) and alpha-catenin. In normal mammary ducts and acini, intense beta-catenin immunoreactivity was present at the basolateral surfaces of luminal epithelium and weak immunoreactivity was observed at the lateral borders of myoepithelial cells. No beta-catenin was revealed at the myoepithelial basal surface. The intercellular expression of beta-catenin, as well as of E-CD and alpha-catenin, was also observed in carcinoma tissues with varying staining intensity. Almost all of 10 intraductal carcinomas and approximately 70% of 41 invasive ductal carcinomas expressed the three molecules at the same level as in normal glands, whereas approximately 80% of 13 invasive lobular carcinomas showed severe deficiency of them. Two lobular carcinomas in situ showed complete absence of all of the proteins. Some of these findings were confirmed biochemically by immunoblotting analysis. In invasive ductal carcinomas, alpha-catenin was reduced more frequently in diffuse than in solid type tumours, whereas the level of expression of beta-catenin and E-CD was unchanged between them. No correlation was present between reduced expression of the adhesion molecules and lymph node metastasis.

Expression of epithelial cadherin and alpha- and beta-catenins in nontumoral livers and hepatocellular carcinomas.

Expression of the cell adhesion molecule, epithelial cadherin (E-CD) and its binding proteins, alpha- and beta-catenins, in normal liver, chronic liver diseases, and hepatocellular carcinomas (HCCs) was investigated immunohistologically. In normal liver, weak immunostaining of E-CD and catenins was observed at the lateral membranes of the hepatocytes, whereas at the interlobular bile duct epithelia, they stained strongly. No immunoreactions were seen in sinusoidal Kupffer cells. Similar results were observed in the majority of livers from chronic hepatitis and cirrhosis sufferers; however, hepatocytes undergoing regeneration and rosette formation, as well as Hering canals and proliferating ductules, showed markedly increased molecular expression. Analysis of 66 HCC lesions revealed that the majority (64.3-96.6%) of thin trabecular- and pseudoglandular-type tumors preserved or overexpressed E-CD and catenins, whereas thick trabecular-type HCCs frequently showed low E-CD and alpha-catenin expression (56.5-65.2% reduction), suggesting that the thick trabecular histology represented diffuse tumor cell growth. Likewise, the E-CD and catenin expression levels correlated with the HCC cell differentiation grades. These collective results indicate that intercellular adhesion mediated by the E-CD-catenin system plays a role in morphological changes in nonmalignant and malignant hepatic diseases.

Establishment of inbred strain of long-haired golden hamster.

Features of a long-haired mutant and inheritance of mutation were investigated in the golden hamster (Mesocricetus auratus). The hair of adult long-haired hamsters was longest at the rump and flanks (approximately 70 mm) and was also unusually long at the neck (45 to 50mm). The hair length of males was more striking than that of females. Mating experiments indicated that an autosomal recessive gene is responsible for the inheritance of long hair. An albino long-haired hamster strain was established by continuous full-sib mating from F2 hybrids, descendants of (acromelanic albino x agouti long-haired) F1 progeny, and genetic homogeneity was confirmed by DNA fingerprinting. The long-haired strain was characterised by the fact that males grew larger than females, in contrast to the other strains of hamsters.

Studies on teratological testing using chicken embryos--effects of solvents, injection sites and the age of the embryo.

The purpose of this study was to examine the effects of solvents, injection sites and embryo age when using chicken embryos for teratological testing. The results obtained were as follows: 1) Solvents: distilled water, physiological saline, sesame oil, 25% ethanol, 0.5% carboxymethylcellulose and 0.1% methylcellulose solution were not toxic in Day-4 embryos (eggs incubated for 4 days). 2) With 6-aminonicotinamide, air space injection more effectively induced malformations in chicken embryos. With boric acid, however, yolk sac injection was better. It was shown therefore that the appropriate injection site varied according to the test drug. 3) 6-aminonicotinamide induced characteristic malformations when injected into embryos of various ages ranging from 4 to 13 days of incubation. On the other hand, boric acid was teratogenetic only when injected into Day-3 or Day-4 embryos. It seems, therefore, that the age of the embryo at the time of administration is of critical importance and that the optimum time of administration varies according to the test drug.

[[Studies on cadmium toxicity in early development of mouse ova. ]].

The purpose of the present experiments was to examine the effect of cadmium on mouse germ cells when this substance was given subcutaneously to the animal in the second meiotic metaphase. Furthermore, its mechanism of action was investigated on the basis of in vitro cultivation, enzymic histochemistry and residue analysis. The progress of gestation after implantation was also followed up. The results obtained were as follows. 1) When cadmium was administered to mice at dose levels of 0.075, 0.15, 0.3, 0.6, 1.2 and 2.4 mg/kg, the fertilization rate (2-cell cleavage rate) was found to be normal at 36 hours after ovulation, but the ratio of normal ova decreased at 84 hours in proportion to increase in the cadmium dose. 2) The ratio of normal ova was 59.9, 39.7 and 11.1% at 38, 62 and 86 hours after ovulation These findings indicate that, during the process of oval growth from the 2-cell to blastocyst stage, degeneration of the ova was gradually induced. 3) To study the developmental ability of the ova as affected by cadmium, 2-cell ova obtained from cadmium-treated mice were cultured in a chemically defined medium. As a result, about 87.0% of the ova ceased to grow by the end of 48 hours of cultivation. 4) Two-cell ova obtained from mice with no cadmium treatment were cultured in a medium containing cadmium. It was found that oval growth was hampered at 4 ppm and over of the substance, but not at 2 ppm. 5) Following administration of 5 mg/kg cadmium, its content in the uterus and ovary was determined. The mean cadmium content was 1.58 +/- 0.7 ppm for the former and 2.42 +/- 1.4 ppm for the latter. In view of the above findings, it was clear that the oval fragility did not result from cadmium in the environment where the embryos grew up. 6) Following administration of 0.15 mg/kg cadmium, all mice were sacrificed on day 18 of gestation to examine the progress of gestation. As a result, the ratio of post- and pre-implantation loss was significantly high as compared to the control. 7) One-cell and 2-cell ova from cadmium-treated mice were examined by means of enzymic histochemistry. Although no abnormality was demonstrated in the 1-cell ova, SDH and NADH2-DH were markedly reduced in the 2-cell ova. NADH2-DH, in particular, decreased quickly from 38 post-ovulative hours onwards.