Overexpression of E2F-5 correlates with a pathological basal phenotype and a worse clinical outcome.

The purpose of the present study is to identify genes that contribute to cell proliferation or differentiation of breast cancers independent of signalling through the oestrogen receptor (ER) or human epidermal growth factor receptor 2 (HER2). An oligonucleotide microarray assayed 40 tumour samples from ER(+)/HER2(-), ER(+)/HER2(+), ER(-)/HER2(+), and ER(-)/HER2(-) breast cancer tissues. Quantitative reverse transcriptase PCR detected overexpression of a cell cycle-related transcription factor, E2F-5, in ER-negative breast cancers, and fluorescence in situ hybridisation detected gene amplification of E2F-5 in 5 out of 57 (8.8%) breast cancer samples. No point mutations were found in the DNA-binding or DNA-dimerisation domain of E2F-5. Immunohistochemically, E2F-5-positive cancers correlated with a higher Ki-67 labelling index (59.5%, P=0.001) and higher histological grades (P=0.049). E2F-5-positive cancers were found more frequently in ER(-)/progesterone receptor (PgR)(-)/HER2(-) cancer samples (51.9%, P=0.0049) and in breast cancer samples exhibiting a basal phenotype (56.0%, P=0.0012). Disease-free survival in node-negative patients with E2F-5-positive cancers was shorter than for patients with E2F-5-negative cancers. In conclusion, we identify, for the first time, a population of breast cancer cells that overexpress the cell cycle-related transcription factor, E2F-5. This E2F-5-positive breast cancer subtype was associated with an ER(-)/PgR(-)/HER2(-) status, a basal phenotype, and a worse clinical outcome.

Observation of beta-ray spectra after penetrating absorbing materials.

beta-Ray spectra after penetrating absorbing materials of various thicknesses were observed by the use of a scintillation-type beta-ray spectrometer equipped with a flat NE-102 plastic scintillator of 5mm thickness for sources of (60)Co, (90)Sr-(90)Y, (137)Cs, (147)Pm and (204)Tl. Although the spectra changed rapidly with increasing absorber thickness, the average beta-ray energy was kept nearly constant for a wider range. These results are consistent in that the beta-ray absorption curve becomes quasi-linear in a semi-logarithmic plot. Spectra including scattered beta-rays from several materials placed behind the source were also measured for (137)Cs and (204)Tl. It may be concluded that mean energy measurements by the use of beta-ray spectrometer of this kind is useful for the identification of nuclides in radiation protection purposes even in worse source-conditions.

Primary structure, expression and localization of two intermediate subunit lectins of Entamoeba dispar that contain multiple CXXC motifs.

We have recently identified 2 surface proteins in Entamoeba histolytica as intermediate subunits of galactose- and N-acetyl-D-galactosamine-inhibitable lectin (EhIgl1 and EhIgl2); these proteins both contain multiple CXXC motifs. Here, we report the molecular characterization of the corresponding proteins in Entamoeba dispar, which is neither pathogenic nor invasive. Two Igl genes encoding 1110 and 1106 amino acids (EdIgl1 and EdIgl2) were cloned from 2 strains of E. dispar. The amino acid sequence identities were 79% between EdIgl1 and EdIgl2, 75-76% between EdIgl1 and EhIgl1, and 73-74% between EdIgl2 and EhIgl2. However, all the CXXC motifs were conserved in the EdIgl proteins, suggesting that the fold conferred by this motif is important for function. Comparison of the expression level of the Igl genes by real-time RT-PCR showed 3-5 times higher expression of EdIgl1 compared to EdIgl2. Most EdIgl1 and EdIgl2 proteins were co-localized on the surface and in the cytoplasm of trophozoites, based on confocal microscopy. However, a different localization of EdIgl1 and EdIgl2 in intracellular vacuoles and a different level of phenotypic expression of the two Igls were also observed. These results demonstrate that Igls are important proteins even in non-pathogenic amoeba and that Igl1 and Igl2 may possess different functions.

Mucosal T-cell immunoregulation varies in early and late inflammatory bowel disease.

BACKGROUND AND AIMS: Crohn's disease is a life-long form of inflammatory bowel disease (IBD) mediated by mucosal immune abnormalities. Understanding of the pathogenesis is limited because it is based on data from adults with chronic Crohn's disease. We investigated mucosal T-cell immunoregulatory events in children with early Crohn's disease. METHODS: Mucosal biopsies and T-cell clones were derived from children experiencing the first attack of Crohn's disease, children with long-standing Crohn's disease, infectious colitis, and children without gut inflammation. RESULTS: As in acute infectious colitis, interleukin (IL) 12 induced T cells from early Crohn's disease to acquire a strongly polarised T helper (Th) type 1 response characterised by high IFN-gamma production and IL12Rbeta2 chain expression. Th1 polarisation was not induced in clones from late Crohn's disease. Mucosal levels of IL12p40 and IL12Rbeta2 messenger RNA were significantly higher in children with early than late Crohn's disease. These results demonstrate that susceptibility to IL12-mediated modulation is strongly dependent on the stage of Crohn's disease. CONCLUSIONS: At the onset of Crohn's disease mucosal T cells appear to mount a typical Th1 response that resembles an acute infectious process, and is lost with progression to late Crohn's disease. This suggests that mucosal T-cell immunoregulation varies with the course of human IBD. Patients with the initial manifestations of IBD may represent an ideal population in which immunomodulation may have optimal therapeutic efficacy.

Definitive experimental evidence for two-band superconductivity in MgB2.

The superconducting-gap of MgB2 has been studied by high-resolution angle-resolved photoemission spectroscopy. The results show that superconducting gaps with values of 5.5 and 2.2 meV open on the sigma band and the pi band, respectively, but both the gaps close at the bulk transition temperature, providing a definitive experimental evidence for the two-band superconductivity with strong interband pairing interaction in MgB2. The experiments validate the role of k-dependent electron-phonon coupling as the origin of multiple-gap superconductivity as well as the high transition temperature of MgB2.

Spinal epidural metastasis from pineal germinoma.

A 16-year-old boy first presented with a pineal tumour identified by neuroimaging but without positive serum or cerebrospinal fluid markers. The tumour disappeared after 50 Gy cranial irradiation. One year later he returned with spinal epidural metastasis from the pineal germinoma and required emergency surgery. Intraoperative findings showed that the spinal tumour had originated from cerebrospinal fluid dissemination and had passed through the spinal nerve sleeve. The pathologic diagnosis of the tumour was of a pure germinoma metastasis. An epidural tumour frequently requires emergency diagnosis and treatment. Attention should be paid to the possibility of this rare but serious clinical situation caused by a metastasis from a pineal germinoma.

A genetic and physical map of the region containing PLASTOCHRON1, a heterochronic gene, in rice ( Oryza sativa L.).

The rice heterochronic gene plastochron1, pla1, shows shorter plastochron and ectopic expression of the vegetative program during the rice reproductive phase resulting in aberrant panicle formation. A genetic and physical map was constructed to isolate the causal gene for the pla1 syndrome. Small-scale mapping was carried out to determine the approximate map position of the pla1 locus, and then a high-resolution genetic map was made for pla1-1, one of the pla1 alleles, using an F(2) population comprising 578 pla1-1 homozygous plants. In a high-resolution genetic map, the pla1-1 locus was found to map between RFLP markers C961 and R1738A on chromosome 10, within a 3.6-cM genetic distance. A physical map encompassing the pla1-1 locus was constructed by overlapping Bacterial Artificial Chromosome (BAC) clones through chromosome walking. PCR-based RFLP markers from BAC-end clones were developed and mapped relative to the pla1 locus. Physical map construction using BAC clones indicated that a BAC clone, B44A10 (167-kb), contained the pla1 locus within 74-kb corresponding to a 0.52-cM genetic distance. Gene prediction of 74-kb region carrying the pla1 locus suggested several candidate genes for the pla1 gene. Identification of a candidate gene for pla1 will be made by sequence analysis of allele variation and cDNA screening.

Characterization of tissue- and cell-type-specific expression of a novel human septin family gene, Bradeion.

Expression changes in subsets of genes occur in the course of altering cell fates, i.e., aging, cell death, and carcinogenesis. These changes simultaneously provide the good candidate as a biomarker for monitoring cancer. We have identified a novel human septin family gene, Bradeion, from adult brain cDNA library by a monoclonal antibody CE5. Northern blot and in situ hybridization analysis showed that Bradeion has two distinct transcripts, approximately 2.2 and 1.7 kb length (alpha and beta, respectively) mainly in brain and slightly in heart, and no expression in any fetal organs. Haplotype analysis placed the gene location at 17q23. The gene contains GTPase motifs highly conserved in the septin family genes that are essential for cytokinesis and cell separation. The transcript of beta form lacks a hydrophobic region, which suggests that this form arises from a single Bradeion gene through unique RNA splicing. Interestingly, this brain-specific Bradeion gene is also expressed in two human cancers, colorectal cancer and malignant melanoma. Ectopic expression of normal Bradeion alpha and beta transcripts were confirmed both in patients' tumor samples and in in vitro cultured human cancer cell lines. Thus the Bradeion provides valuable tools as a tumor-specific and selective marker.

Decreased Bax expression by mucosal T cells favours resistance to apoptosis in Crohn's disease.

BACKGROUND: Activated T cells are more susceptible to apoptosis than resting T cells. As intestinal T cells normally exhibit a higher state of activation, increased apoptosis may be necessary to maintain immune homeostasis in the specialised microenvironment of the mucosa. On the other hand, in Crohn's disease (CD) mucosal T cells are resistant to apoptosis, suggesting abnormal regulation of cell death mechanisms. AIMS: To investigate differences in expression of anti- and proapoptotic Bcl-2 family proteins, key regulators of apoptosis, between circulating and mucosal T cells, and possible alterations in CD. PATIENTS AND METHODS: Lamina propria T cells (LPT) were isolated from 10 control, seven CD, and eight ulcerative colitis (UC) patients, and peripheral blood T cells (PBT) from healthy volunteers. Purified T cells were stained intracellularly for Bcl-2, Bcl-x(L), and Bax, and mean fluorescence intensity measured by flow cytometry. RESULTS: Compared with PBT, the expression level of Bcl-2 and Bax, but not Bcl-x(L), was significantly greater in LPT, resulting in lower Bcl-x(L)/Bax ratios. In PBT, Bax expression was highly and significantly correlated with both Bcl-2 and Bcl-x(L), but correlation with Bcl-2 was absent in LPT. Bax expression in CD, but not UC, LPT was significantly lower than in control LPT, resulting in a significantly higher Bcl-x(L)/Bax ratio. The significant correlation of Bcl-x(L) to Bax was preserved in CD, but not UC, LPT. CONCLUSIONS: Regulation of Bcl-2 family protein expression differs between circulating and mucosal T cells, probably underlying diverse survival potentials. In CD LPT, a low Bax expression and a high Bcl-x(L)/Bax ratio favour resistance to apoptosis and may contribute to the chronicity of inflammation.

Histopathological analyses of silent pituitary somatotroph adenomas using immunohistochemistry, in situ hybridization and confocal laser scanning microscopic observation.

To characterize the morphological and functional aspects of silent somatotroph adenomas with paradoxical responses of GH in TRH or GnRH provocation tests, which are considered to be a useful strategy for endocrinological identification of silent somatotroph adenomas, we examined three silent somatotroph adenomas histopathologically. The adenomas were investigated by immunohistochemistry, including the highly sensitive catalyzed signal amplification system, the non-radioisotopic in situ hybridization method, and confocal laser scanning microscopy. GH production and GH-immunopositive secretory granules in the adenoma cells were demonstrated histopathologically, and the adenomas were interpreted as being densely granulated somatotroph adenomas. Endocrinological identification of silent somatotroph adenomas in combination with paradoxical responses of GH in TRH or GnRH provocation tests may elucidate the increasing number of silent somatotroph adenomas that have been regarded as mammotroph or clinically inactive adenomas. One should be aware of the differences between the previously reported silent somatotroph adenomas, most of which are sparsely granulated somatotroph adenomas, a somatotroph adenomas with paradoxical and the silent somatotroph adenomas, most of which are sparsely granulated somatotroph adenomas, and the silent somatotroph adenomas with paradoxical responses of GH in TRH or GnRH provocation tests, which are densely granulated somatotroph adenomas.

Three-dimensional imaging of hormone-secreting cells and their microvessel environment in estrogen-induced prolactinoma of the rat pituitary gland by confocal laser scanning microscopy.

This study focused on the three-dimensional imaging of hormone-secreting cells and their microvascular environment in estrogen-induced prolactinoma of the rat pituitary gland. Adult female Wistar-Imamichi rats were injected with estradiol dipropionate and killed 7 weeks later. Some rats given estrogen for 7 weeks also were injected with bromocriptine before killing. To obtain a detailed three-dimensional image of microvessels, dialyzed fluorescein isothiocyanate (FITC)-conjugated gelatin was injected into the left ventricle of the rat heart. After the perfusion, the pituitary glands were resected and subjected to immunohistochemistry (IHC). To evaluate the effects of estrogen and bromocriptine, IHC was performed with antibodies against prolactin (PRL), adrenocorticotropic hormone (ACTH), and growth hormone (GH). With the combination, microvessels and cells containing PRL, ACTH, and GH could be clearly identified by confocal laser scanning microscopy (CLSM). The PRL cells increased in number and became hypertrophic after prolonged exposure to estrogen. With bromocriptine administration after estrogen treatment, however, PRL cells decreased in number and became atrophic. The current study revealed that estrogen and bromocriptine had significant effects on PRL secretion and the microvascular environment. Therefore, this technique (FITC injection and IHC) with CLSM is suitable for the three-dimensional imaging of hormone-secreting mechanisms under various conditions.

Effect of calcium in assay medium on D value of Bacillus stearothermophilus ATCC 7953 spores.

The D value of commercial biological indicator spore strips using Bacillus stearothermophilus ATCC 7953 was increased by higher calcium concentrations in assay media. The calcium concentration in assay media varied among the manufacturers. The calcium concentration in assay media is an important factor to consider to minimize the variation of D value.

Shoot organization genes regulate shoot apical meristem organization and the pattern of leaf primordium initiation in rice.

The mechanism regulating the pattern of leaf initiation was analyzed by using shoot organization (sho) mutants derived from three loci (SHO1, SHO2, and SHO3). In the early vegetative phase, sho mutants show an increased rate of leaf production with random phyllotaxy. The resulting leaves are malformed, threadlike, or short and narrow. Their shoot apical meristems are relatively low and wide, that is, flat shaped, although their shape and size are highly variable among plants of the same genotype. Statistical analysis reveals that the shape of the shoot meristem rather than its size is closely correlated with the variations of plastochron and phyllotaxy. Rapid and random leaf production in sho mutants is correlated with the frequent and disorganized cell divisions in the shoot meristem and with a reduction of expression domain of a rice homeobox gene, OSH1. These changes in the organization and behavior of the shoot apical meristems suggest that sho mutants have fewer indeterminate cells and more determinate cells than wild type, with many cells acting as leaf founder cells. Thus, the SHO genes have an important role in maintaining the proper organization of the shoot apical meristem, which is essential for the normal initiation pattern of leaf primordia.

Plasma lactate concentration and muscle blood flow during dynamic exercise with negative-pressure breathing.

This study assessed the hypothesis that increasing cardiac filling pressure (CFP) would enhance contracting muscle blood flow (MBF) by stretching cardiopulmonary baroreceptors and attenuate the increase in plasma lactate concentration ([Lac(-)](p)) during dynamic exercise. Continuous negative-pressure breathing (CNPB) (-15 cmH(2)O) was used to increase the CFP by accelerating the venous return to the heart. In the first series of experiments, 10 men performed a graded exercise seated on a cycle ergometer with (N1) and without CNPB (C1). The increase in [Lac(-)](p) for N1 was attenuated at 60%, 90%, and 100% of maximal exercise intensity compared with that in C1 (P < 0.001). Also, the increases in mean arterial pressure (MAP) and plasma catecholamine concentrations were attenuated in N1 compared with those in C1 throughout the graded exercise (P < 0.05). However, heart rate and pulse pressure were not significantly influenced by CNPB. Second, we studied the impact of CNPB on forearm MBF during a rhythmic handgrip exercise in 5 of the 10 subjects. Forearm MBF was measured immediately after cessation of the exercise by venous occlusion plethysmography at rest, 30%, 50%, and 70% of maximal work load (WL(max)) with (N2) and without CNPB (C2). Forearm MBF and vascular conductance for both trials increased with the increase in intensity, but forearm skin blood flow measured by laser-Doppler flowmetry remained unchanged. MBF and vascular conductance in N2, however, increased more than in C2 at every intensity (P < 0.01) except for MBF at 70% WL(max), whereas the increase in MAP for N2 was attenuated compared with that in C2 (P < 0.05). Thus augmented active muscle vasodilation occurred in N2 with a lower increase in MAP compared with that in C2. These findings suggest that the stretch of intrathoracic baroreceptors, such as cardiopulmonary mechanoreceptors, by CNPB increased MBF by suppressing sympathetic nerve activity. The attenuation of the increase in [Lac(-)](p) might be caused, at least partially, by the increased MBF.

Zinc distribution in the small-intestinal digesta of pigs fed skim milk powder or defatted soybean flour.

Zinc distribution in the small-intestinal digesta was studied in pigs fed a skim milk powder (SMP)-based or defatted soybean flour (DSF)-based diet. Ten pigs were fed experimental diets for 30 d. Serum and femoral zinc concentrations were lower in the DSF group than in the SMP group, suggesting the lower zinc availability in the DSF diet than in the SMP diet. In the digesta of small intestine, zinc solubility was higher in the SMP group than in the DSF group. A gel permeation high-performance liquid chromatography showed that the SMP group exhibited four zinc peaks in the chromatogram of the supernatant of digesta. The chromatogram in the DSF group showed four zinc peaks corresponding to those of the SMP group. However, the first zinc peak was smaller and the second peak was larger in the DSF group than in the SMP group. Dietary treatment did not affect the other peaks of zinc. Although the high solubility of zinc in the digesta of SMP group is considered to result in the high availability of zinc, the difference of zinc distribution in the liquid phase of digesta may partly affect zinc availability.

Differentiation of necrotic cell death with or without lysosomal activation: application of acute liver injury models induced by carbon tetrachloride (CCL4) and dimethylnitrosamine (DMN).

We investigated the relationship between DNA degradation and lysosome activity (loss of lysosomal integrity) in necrotic cell death induced by carbon tetrachloride (CCl4) and dimethylnitrosamine (DMN): coagulation necrosis and hemorrhagic necrosis, respectively. TdT-mediated dUTP-biotin nick end-labeling (TUNEL) and enzyme histochemistry for acid phosphatase were performed in both models and results were analyzed by light microscopy, electron microscopy, and confocal laser scanning microscopy (CLSM). In the CCl(4)-injected liver, TUNEL staining was closely associated with release of lysosomal enzymes into the cytoplasm, and intranuclear deposition of lysosomal enzymes took place at an early stage of subcellular damage. In the DMN-injected liver, TUNEL-positive nuclei tended to have well-preserved lysosomes and centrally localized TUNEL signals. It was assumed that acute hepatocellular damage in the CCl4-injected liver would be characterized by necrotic cell death with lysosome activation and that damage in the DMN-injected liver would be necrotic cell death without lysosome activation. In the DMN-injected liver, DNA degradation may be selectively induced in the nuclear center, in which heterochromatin (including inactive chromatin) is believed to be a target. We concluded that necrotic cell death, i.e., DNA degradation, would be at least divided into two types, with/without association with lysosome activation, represented by necrotic cell death in the CCl4-injected liver and that in the DMN-injected liver.

Lecithinized copper, zinc-superoxide dismutase ameliorates prolonged hypoxia-induced injury of cardiomyocytes.

Recent studies have suggested that prolonged hypoxia results in increased production of reactive oxygen species in cardiomyocytes, which leads to apoptosis of these cells. We previously showed that lecithinized recombinant human copper, zinc-superoxide dismutase (rhSOD) showed increased bioavailability through greater membrane affinity and a longer half-life than unmodified SOD. The purpose of this study was to investigate whether lecithinized SOD plays a protective role against hypoxic injury in cardiomyocytes. Cultured rat cardiomyocytes incubated with lecithinized SOD (100 U/ml), unmodified SOD (100 U/ml), or vehicle alone were subjected to hypoxia for up to 72 h. Lecithinized SOD, but not unmodified SOD, was successfully delivered intracellularly, which was verified by Western blot and confocal laser-scanning microscopy. Treatment of cells with lecithinized SOD significantly suppressed hypoxia-induced cell damage. Since lecithinized SOD also suppressed hypoxia-induced DNA fragmentation, the improved cell survival provided by lecithinized SOD is thought to be mediated by its antiapoptotic effect. In summary, lecithinization resulted in a facilitated rhSOD delivery into cultured cardiomyocytes, which reduced mortality of cardiomyocytes exposed to prolonged hypoxia.

Validation of D value by different SCD culture medium manufacturer and/or different SCD culture medium constituent.

Variation in decimal reduction (D) time/value for attaining sterility assurance was reported in terms of difference between lot to lot and/or manufacturer to manufacturer of soybean casein digest (SCD) culture medium. The D value variation phenomenon itself due to SCD culture medium was reported by Graham et al; however, it was not sufficiently clarified what constituent(s) of SCD culture medium would mainly contribute to the variation of D value. If it is not well clarified, the reproducible sterility assurance cannot be successfully attained. The authors studied individually the constituent of SCD broth (SCDB), SCD agar (SCDA) and SCD broth plus 1.5% agar (SCDB/A). As a result, the culture medium constituent causing D value variation among SCD culture medium was determined as the difference of calcium (Ca) amount as well as the presence and the absence of K2HPO4 in SCD culture medium. Additionally, it was clarified D value differed significantly between SCDB and SCDA. D value using the former was around 1 min lower than that using the latter. This phenomenon can be explained from the formation of insoluble CaHPO4 in SCDB culture medium, resulting in insufficiency of usable Ca to B. stearothermophilus in SCDB culture medium.

Three-dimensional imaging of tumor angiogenesis.

OBJECTIVE: To three-dimensionally visualize the microvessel environment of tumor angiogenesis by confocal laser scanning microscopy (CLSM). STUDY DESIGN: To reveal underlying mechanisms of tumor angiogenesis, a 7, 12-dimethylbenz(a) anthracene-induced rat cancer model was used. For demonstrating tumor vasculature, fluorescence injection method (FITC-conjugated gelatin solution) was employed. FITC gelatin was injected into the left ventricle of the rat heart. After complete perfusion, the mammary glands were resected, fixed under ice cold conditions and subjected to immunohistochemistry (IHC) for tumor cells. The LSM-410 (Carl Zeiss, Jena, Germany) was employed on thick sections (300-2,000 microns) to elucidate detailed microvessel networks (MVN) and tumor cells. RESULTS: Tumor vasculature on thick sections was clearly detected by CLSM at the maximum focus depth of 2,000 microns. Three-dimensional (3-D), reconstructed images of normal mammary glands showed regular and linear MVN. In DMBA-induced mammary cancer, vascular density of MVN was markedly increased and showed an anastomosing, irregular MVN pattern. Furthermore, focal segmentation and tortuous, branching patterns of microvessels were also seen. CONCLUSION: Application of the fluorescence injection method and IHC using CLSM was very useful for studying the 3-D relationship between tumor angiogenesis and neoplastic epithelial changes. These results suggest that application of this technique is ideal for studying 3-D imaging of tumor angiogenesis.