Temperature regulation during rest and exercise in the cold in premenarcheal and menarcheal girls.

Temperature regulation during exercise in the cold was examined in 13 adolescent female individuals, aged 13-18 yr. Six girls with established menstrual cycles comprised the eumenorrheic menarcheal (EM) group, and seven nonmenstruating girls comprised the premenarcheal (PM) group. During the first visit, maximal oxygen consumption (Vo(2 max)), height, weight, and percent body fat were measured. The second visit included a determination of metabolic rate in thermoneutrality (21 degrees C), consisting of a 10-min rest period and 20 min of cycling (30% of Vo(2 max)), and a cold test (5 degrees C, 40% humidity, <0.3 m/s air velocity) involving a 20-min rest period and 40 min of cycling (30% of Vo(2 max)). Subjects in the EM group were tested twice in the chamber: once during the follicular and once during the luteal phase. Heat production per kilogram in thermoneutrality and in the cold was significantly (P < 0.05) higher in the PM compared with the EM girls. However, the PM girls had a significantly (P < 0.05) lower core temperature in the cold than the EM group. PM girls also had a significantly higher body surface area-to-mass ratio compared with the EM girls. Although percent body fat between groups was not significantly different, within the PM group percent body fat explained 79% (P < 0.01) of the variance in the decrease of core temperature. There were no menstrual phase-related differences in temperature regulation in either the thermoneutral or cold environment. In conclusion, menstrual phase does not influence temperature regulation in female individuals during adolescence. EM girls had lower metabolic heat production but maintained their core temperature more effectively in the cold than did the PM girls. This thermoregulatory difference between PM and EM girls is mainly a function of geometric differences with maturation-related peripheral vasoconstrictive differences maybe limiting the effectiveness of the mechanism of increased heat storage in younger female individuals.     

Cascade pattern recognition structure for improving quantitative assessment of estrogen receptor status in breast tissue carcinomas

OBJECTIVE: To develop and validate a computer-based approach for the quantitative assessment of estrogen receptor (ER) status in breast tissue specimens for breast cancer management. STUDY DESIGN: Microscopy images of 32 immunohistochemically (IHC) stained specimens of breast cancer biopsies were digitized and were primarily assessed for ER status (percentage of positively stained nuclei) by a histopathologist. A pattern recognition system was designed for automatically assessing the ER status of the IHC-stained specimens. Nuclei were automatically segmented from background by a pixel-based unsupervised clustering algorithm and were characterized as positively stained or unstained by a supervised classification algorithm. This cascade structure boosted the system's classification accuracy. RESULTS: System performance in correctly characterizing the nuclei was 95.48%. When specifying each case's ER status, system performance was statistically not significantly different to the physician's assessment (p = 0.13); when ranking each case to a particular 5-scale ER-scoring system (giving the chance of response to endocrine treatment), the system's score and the physician's score were in agreement in 29 of 32 cases. CONCLUSION: The need for reliable and operator independent ER-status estimation procedures may be served by the design of efficient pattern recognition systems to be employed as support opinion tools in clinical practice.     

Engineering sensitive glutathione transferase for the detection of xenobiotics

Cytosolic glutathione transferases (GSTs) are a major reserve of high-capacity ligand binding proteins which recognise a large variety of hydrophobic compounds. In the present study, the binding of non-substrate xenobiotic compounds (herbicides and insecticides) to maize GST I was investigated by employing kinetic inhibition studies, site-directed mutagenesis and molecular modelling studies. The results showed that the xenobiotics bind at the substrate binding site. Based on in silico docking analysis, two residues were selected for assessing their contribution to xenobiotic binding. The mutant Gln53Ala of GST I Exhibits 9.2-fold higher inhibition potency for the insecticide malathion, compared to the wild-type enzyme. A potentiometric assay was developed for the determination of malathion using the Gln53Ala mutant enzyme. The assay explores the ability of the xenobiotic to promote inhibition of the GST-catalysing 1-chloro-2,4-dinitrobenzene (CDNB)/glutathione (GSH) conjugation reaction. The sensing scheme is based on the pH change occurring in a low buffer system by the GST reaction, which is measured potentiometrically using a pH electrode. Calibration curve was obtained for malathion, with useful concentration range 0-20muM. The method's reproducibility was in the order of +/-3-5% and malathion recoveries were 96.7+/-2.8%. Immobilized Gln53Ala mutant GST was used to assemble a biosensor for malathion. The enzyme was immobilized by crosslinking with glutaraldehyde and trapped behind a semipermeable membrane in front of the pH electrode. The results demonstrated that the immobilized enzyme behaved similar to free enzyme.     

CRD-BP/IMP1 expression characterizes cord blood CD34+ stem cells and affects c-myc and IGF-II expression in MCF-7 cancer cells

The coding region determinant-binding protein/insulin-like growth factor II mRNA-binding protein (CRD-BP/IMP1) is an RNA-binding protein specifically recognizing c-myc, leader 3' IGF-II and tau mRNAs, and the H19 RNA. CRD-BP/IMP1 is predominantly expressed in embryonal tissues but is de novo activated and/or overexpressed in various human neoplasias. To address the question of whether CRD-BP/IMP1 expression characterizes certain cell types displaying distinct proliferation and/or differentiation properties (i.e. stem cells), we isolated cell subpopulations from human bone marrow, mobilized peripheral blood, and cord blood, all sources known to contain stem cells, and monitored for its expression. CRD-BP/IMP1 was detected only in cord blood-derived CD34(+) stem cells and not in any other cell type of either adult or cord blood origin. Adult BM CD34(+) cells cultured in the presence of 5'-azacytidine expressed de novo CRD-BP/IMP1, suggesting that epigenetic modifications may be responsible for its silencing in adult non-expressing cells. Furthermore, by applying the short interfering RNA methodology in MCF-7 cells, we observed, subsequent to knocking down CRD-BP/IMP1, decreased c-myc expression, increased IGF-II mRNA levels, and reduced cell proliferation rates. These data 1) suggest a normal role for CRD-BP/IMP1 in pluripotent stem cells with high renewal capacity, like the CB CD34(+) cells, 2) indicate that altered methylation may directly or indirectly affect its expression in adult cells, 3) imply that its de novo activation in cancer cells may affect the expression of c-Myc and insulin-like growth factor II, and 4) indicate that the inhibition of CRD-BP/IMP1 expression might affect cancer cell proliferation.     

A HER-2/neu peptide admixed with PLA microspheres induces a Th1-biased immune response in mice

The elimination of cancer cells requires strong cellular immune responses, and these responses are induced by the activation of Th1 lymphocytes. In this work, the possibility of inducing a Th1 type of immune response in vivo by mixing a HER-2/neu synthetic CTL (cytotoxic T lymphocyte) peptide [HER-2/neu (789-797)], with poly-lactide (PLA) microspheres was investigated. Various formulations of the peptide were administered to HLA-A2.1 transgenic (HHD) mice. Cellular experiments, assessing proliferation and cytokine determination in splenocyte culture supernatants, were carried out in order to evaluate the type of immune response to the antigen. The in vivo administration of the peptide antigen admixed with the PLA microspheres induced a potent immune response which was comparable to that induced by the combination of the antigen in complete Freund's adjuvant (CFA). Furthermore, the cytokine profile produced by the T lymphocytes of the immunized animals indicated that the combination of the peptide antigen with the PLA microspheres induced a strong Th1 biased immune response to the antigen. The time of peptide incubation with the microspheres prior to administration did not affect the immune response, which further simplifies the preparation of this type of vaccine. The results justify further investigation of the possibility of inducing effective cellular immune responses against cancer cells overexpressing HER-2/neu molecules by simply mixing appropriate HER-2/neu peptide antigens with PLA microspheres.     

Human trabecular bone-derived osteoblasts support human osteoclast formation in vitro in a defined, serum-free medium

While it has been assumed that osteoblasts in the human support osteoclast formation, in vitro evidence of this is currently lacking. We tested the ability of normal human trabecular bone-derived osteoblasts (NHBCs) to support osteoclast formation from human peripheral blood mononuclear cells (PBMC) in response to treatment with either 1alpha,25-dihydroxyvitamin D3 (1,25D) or parathyroid hormone (PTH), using a serum-replete medium previously used to support human osteoclast formation on a stroma of murine ST-2 cells. Under these conditions, NHBC did not support osteoclast formation, as assessed by morphological, histochemical, and functional criteria, despite our previous results demonstrating a link between induction of RANKL mRNA expression and NHBC phenotype in these media. We next tested a defined, serum-free medium (SDM) on NHBC phenotype, their expression of RANKL and OPG, and their ability to support osteoclast formation. SDM, containing dexamethasone (DEX) and 1,25D, induced phenotypic maturation of NHBC, based on the expression of STRO-1 and the bone/liver/kidney isoform of alkaline phosphatase (AP). PTH as a single factor did not induce phenotypic change. 1,25D and DEX induced the greatest ratio of RANKL:OPG mRNA, predictive of supporting osteoclast formation. Consistent with this, co-culture of NHBC with CD14+ PBMC, or bone marrow mononuclear cell (BMMC), or CD34+ BMMC precursors in SDM + 1,25D + DEX, resulted in functional osteoclast formation. Osteoclast formation also occurred in PTH + DEX stimulated co-cultures. Interestingly, SDM supplemented with recombinant RANKL (25-100 ng/ml) and M-CSF (25 ng/ml), did not induce osteoclast formation from any of the osteoclast precursor populations in stromal-free cultures, unlike serum-replete medium. This study demonstrates that under the appropriate conditions, adult human primary osteoblasts can support de novo osteoclast formation, and this model will enable the detailed study of the role of both cell types in this process.     

SCHIZORIZA controls an asymmetric cell division and restricts epidermal identity in the Arabidopsis root

The primary root of Arabidopsis has a simple cellular organisation. The fixed radial cell pattern results from stereotypical cell divisions that occur in the meristem. Here we describe the characterisation of schizoriza (scz), a mutant with defective radial patterning. In scz mutants, the subepidermal layer (ground tissue) develops root hairs. Root hairs normally only form on epidermal cells of wild-type plants. Moreover, extra periclinal divisions (new wall parallel to surface of the root) occur in the scz root resulting in the formation of supernumerary layers in the ground tissue. Both scarecrow (scr) and short root (shr) suppress the extra periclinal divisions characteristic of scz mutant roots. This results in the formation of a single layered ground tissue in the double mutants. Cells of this layer develop root hairs, indicating that mis-specification of the ground tissue in scz mutants is uncoupled to the cell division defect. This suggests that during the development of the ground tissue SCZ has two distinct roles: (1) it acts as a suppressor of epidermal fate in the ground tissue, and (2) it is required to repress periclinal divisions in the meristem. It may act in the same pathway as SCR and SHR.     

Madagascar's grasses and grasslands: anthropogenic or natural?

Grasses, by their high productivity even under very low pCO₂, their ability to survive repeated burning and to tolerate long dry seasons, have transformed the terrestrial biomes in the Neogene and Quaternary. The expansion of grasslands at the cost of biodiverse forest biomes in Madagascar is often postulated as a consequence of the Holocene settlement of the island by humans. However, we show that the Malagasy grass flora has many indications of being ancient with a long local evolutionary history, much predating the Holocene arrival of humans. First, the level of endemism in the Madagascar grass flora is well above the global average for large islands. Second, a survey of many of the more diverse areas indicates that there is a very high spatial and ecological turnover in the grass flora, indicating a high degree of niche specialization. We also find some evidence that there are both recently disturbed and natural stable grasslands: phylogenetic community assembly indicates that recently severely disturbed grasslands are phylogenetically clustered, whereas more undisturbed grasslands tend to be phylogenetically more evenly distributed. From this evidence, it is likely that grass communities existed in Madagascar long before human arrival and so were determined by climate, natural grazing and other natural factors. Humans introduced zebu cattle farming and increased fire frequency, and may have triggered an expansion of the grasslands. Grasses probably played the same role in the modification of the Malagasy environments as elsewhere in the tropics.