Carbon-13 labelling strategy for studying the ATP metabolism in individual yeast cells by micro-arrays for mass spectrometry

Isotopic labelling of cellular metabolites, used in conjunction with high-density micro-arrays for mass spectrometry enables observation of ATP metabolism in single yeast cells.     

Long-term monitoring of fecal steroid hormones in female snow leopards (Panthera uncia) during pregnancy or pseudopregnancy

Knowledge of the basic reproductive physiology of snow leopards is required urgently in order to develop a suitable management conditions under captivity. In this study, the long-term monitoring of concentrations of three steroid hormones in fecal matter of three female snow leopards was performed using enzyme immunoassays: (1) estradiol-17β, (2) progesterone and (3) cortisol metabolite. Two of the female animals were housed with a male during the winter breeding season, and copulated around the day the estradiol-17β metabolite peaked subsequently becoming pregnant. The other female was treated in two different ways: (1) first housed with a male in all year round and then (2) in the winter season only. She did not mate with him on the first occasion, but did so latter around when estradiol-17β metabolite peaked, and became pseudopregnant. During pregnancy, progesterone metabolite concentrations increased for 92 or 94 days, with this period being approximately twice as long as in the pseudopregnant case (31, 42, 49 and 53 days). The levels of cortisol metabolite in the pseudopregnant female (1.35 μg/g) were significantly higher than in the pregnant females (0.33 and 0.24 μg/g) (P<0.05). Similarly, during the breeding season, the levels of estradiol-17β metabolite in the pseudopregnant female (2.18 μg/g) were significantly higher than those in the pregnant females (0.81 and 0.85 μg/g) (P<0.05). Unlike cortisol the average levels of estradiol-17β during the breeding season were independent of reproductive success.The hormone levels may also be related to housing conditions and the resulting reproductive success in female leopards. The female housed with a male during the non-breeding season had high levels of cortisol metabolites and low levels of estradiol-17β in the breeding season, and failed to become pregnant. This indicates that housing conditions in snow leopards may be an important factor for normal endocrine secretion and resulting breeding success.     

Mitochondrial DNA mutations regulate metastasis of human breast cancer cells

Mutations in mitochondrial DNA (mtDNA) might contribute to expression of the tumor phenotypes, such as metastatic potential, as well as to aging phenotypes and to clinical phenotypes of mitochondrial diseases by induction of mitochondrial respiration defects and the resultant overproduction of reactive oxygen species (ROS). To test whether mtDNA mutations mediate metastatic pathways in highly metastatic human tumor cells, we used human breast carcinoma MDA-MB-231 cells, which simultaneously expressed a highly metastatic potential, mitochondrial respiration defects, and ROS overproduction. Since mitochondrial respiratory function is controlled by both mtDNA and nuclear DNA, it is possible that nuclear DNA mutations contribute to the mitochondrial respiration defects and the highly metastatic potential found in MDA-MB-231 cells. To examine this possibility, we carried out mtDNA replacement of MDA-MB-231 cells by normal human mtDNA. For the complete mtDNA replacement, first we isolated mtDNA-less (ρ(0)) MDA-MB-231 cells, and then introduced normal human mtDNA into the ρ(0) MDA-MB-231 cells, and isolated trans-mitochondrial cells (cybrids) carrying nuclear DNA from MDA-MB-231 cells and mtDNA from a normal subject. The normal mtDNA transfer simultaneously induced restoration of mitochondrial respiratory function and suppression of the highly metastatic potential expressed in MDA-MB-231 cells, but did not suppress ROS overproduction. These observations suggest that mitochondrial respiration defects observed in MDA-MB-231 cells are caused by mutations in mtDNA but not in nuclear DNA, and are responsible for expression of the high metastatic potential without using ROS-mediated pathways. Thus, human tumor cells possess an mtDNA-mediated metastatic pathway that is required for expression of the highly metastatic potential in the absence of ROS production.     

Gene selection for classification of cancers using probabilistic model building genetic algorithm

Recently, DNA microarray-based gene expression profiles have been used to correlate the clinical behavior of cancers with the differential gene expression levels in cancerous and normal tissues. To this end, after selection of some predictive genes based on signal-to-noise (S2N) ratio, unsupervised learning like clustering and supervised learning like k-nearest neighbor (k NN) classifier are widely used. Instead of S2N ratio, adaptive searches like Probabilistic Model Building Genetic Algorithm (PMBGA) can be applied for selection of a smaller size gene subset that would classify patient samples more accurately. In this paper, we propose a new PMBGA-based method for identification of informative genes from microarray data. By applying our proposed method to classification of three microarray data sets of binary and multi-type tumors, we demonstrate that the gene subsets selected with our technique yield better classification accuracy.     

<Emphasis Type="Italic">cis</Emphasis>-Cinnamoyl Glucoside as a Major Plant Growth Inhibitor Contained in <Emphasis Type="Italic">Spiraea prunifolia</Emphasis>

Crude extracts of the leaves of Spiraea prunifolia Sieb. showed high plant-growth-inhibiting activity comparable to that of S. thunbergii extracts. To isolate the causal compound in S. prunifolia, we performed bioassay-directed purification by monitoring the biological activity per unit weight of the organism containing the bioactive compound (total activity). We isolated 1-O-cis-cinnamoyl-β-D-glucopyranose (cis-CG) and identified it as the most important growth-inhibiting constituent in the crude extracts. We did not detect 6-O-(4′-hydroxy-2′-methylenebutyroyl)-1-O-cis-cinnamoyl-β-D-glucopyranose (cis-BCG) in S. prunifolia, though it is a major plant growth inhibitor in S. thunbergii together with cis-CG. We estimated the cis-CG content in S. prunifolia to be 3.84 mmol kg⁻¹ F.W. This amount is comparable to the cis-CG plus cis-BCG content in S. thunbergii (3.59 mmol kg⁻¹ F.W.). This indicates that S. prunifolia and S. thunbergii have equally high potential to inhibit plant growth, and cis-CG acts as the most important plant-growth inhibitor in S. prunifolia extracts.     

Homeobox B3, FoxA1 and FoxA2 interactions in epithelial lung cell differentiation of the multipotent M3E3/C3 cell line

HOM/C homeobox (Hox) and forkhead box (Fox) factors are reported to be expressed in the foregut endoderm and are subsequently detected in a spatio-temporal pattern during lung development. Some of these factors were reported to influence the expression of lung marker proteins or to modulate lung development. To clarify the molecular mechanisms for generating functional lung cells from progenitor cell populations, we introduced the forkhead box factors, FoxA1 and FoxA2, and the homeobox factor, HoxB3, into the differentiation process in a multipotent hamster lung epithelial M3E3/C3 cell line. Ectopic expression of FoxA2 promoted differentiation to Clara-like cells with up-regulation of the expression of the lung marker proteins, Clara cell-specific 10-kDa protein and surfactant protein-B. In contrast, FoxA1 repressed the differentiation. HoxB3 transfection induced FoxA2 expression transiently at the pre-differentiation stage. The endogenous HoxB3 expression level decreased at later stages of Clara-like cell differentiation, and the attenuation was enhanced by FoxA2 transfection. HoxB3 is a putative upstream regulator that enhances FoxA2 expression at the pre-differentiation stage. In addition, we found that the expression of HoxA4, HoxA5, and HoxC9 increased differentially during Clara-like cell differentiation. These results suggest that HoxB3 may be a putative positive regulator of FoxA2 expression at the pre-differentiation stage, and those interactions of Fox factors and Hox factors could participate in Clara cell differentiation.     

Structural and functional evolution of three cardiac natriuretic peptides

Natriuretic peptides (NPs) are a group of hormones playing important roles in cardiovascular and osmoregulatory systems in vertebrates. Among the NP subtypes, atrial NP (ANP), B-type NP (BNP), and ventricular NP (VNP) are circulating hormones expressed exclusively in the heart (cardiac NPs). The constitution of cardiac NPs is variable among species of vertebrates. In order to understand the evolutionary and functional significance of such variation, we performed a systematic survey of cardiac NP cDNAs in nine taxonomically diverse teleosts inhabiting environments of varying salinity. The discovery of the coexistence of the ANP, BNP, and VNP genes in the eel and rainbow trout suggested that the ancestral teleost had all three cardiac NPs. As the VNP cDNA was undetectable in ayu and six species of Neoteleostei, it is possible that VNP was lost before the divergence of Osmeroidei. The ANP gene was also undetectable in the medaka. Thus, only the BNP gene is universal in species examined in the present study. Synthetic medaka BNP preferentially activated two medaka GC-A-type receptors, suggesting that the three cardiac NPs share the same receptor. However, the regulation of BNP expression may be the most strict because ATTTA repeats in the 3'-untranslated region and the dibasic motif in the ring are conserved among teleosts and tetrapods. Linkage analyses in the rainbow trout located ANP, BNP, and VNP genes on the same chromosome, which suggested the generation of the VNP gene by tandem duplication as observed with ANP and BNP genes. If the duplication occurred before the divergence of tetrapods and teleosts, VNP may exist in the tetrapod lineage.     

BCG modulation of anaphylactic antibody response, airway inflammation and lung hyperreactivity in genetically selected mouse strains (Selection IV-A)

The effect of Bacillus Calmette-Guérin (BCG) treatment in allergic pulmonary reaction was studied in mice genetically selected accordingly to a High (H-IVA) or Low (L-IVA) antibody responsiveness. Mice were immunized with ovalbumin (OVA) or OVA plus BCG. Two days after nasal antigenic challenge, seric IgE and IgG1 anti-OVA, eosinophils in pulmonary tissue, inflammatory cells in bronchoalveolar lavage and the compliance and conductance of respiratory system were evaluated. H-IVA mice were found more susceptible than L-IVA, and BCG was able to inhibit simultaneously the production of IgE, the bronchopulmonary inflammation and bronchial hyperresponsiveness in these genetically selected mice.     

Osteoblastic activity and estrogenic response in the regenerating scale of goldfish, a good model of osteogenesis

Osteogenesis in the teleost was morphologically observed using regenerating scales of goldfish. Histological observations indicated that osteoblasts around the regenerating scales on days 7 to 10 were greater in size and number than those at other stages. Therefore, further experiments were carried out to examine the activity of osteoblasts in the regenerating period. To quantify their osteoblastic activities, scales on the left side of the body were taken, and the regenerating scales were then used to measure the activities of alkaline phosphatase (ALP), a marker of osteoblasts, on days 7, 10, and 15. The ontogenic scales on the right side of the body were also collected and used to measure ALP activity on the same days. Osteoblasts at all stages of regenerating scales were more active than those in the remaining ontogenic scales. The regenerating scales on day 10 had the highest activity. Furthermore, we found that estrogen receptor (ER) mRNA was expressed in the regenerating scales because estrogen participates in osteoblastic growth and differentiation in mammals. Therefore, using a scale culture system reported previously, the estrogenic response was examined in the ontogenic and regenerating scales on day 10. The reactivity was much higher in regenerating scales, although estrogen treatment significantly activated the osteoblastic activities in both scales. We are the first to demonstrate that ER is expressed in regenerating scales and that estrogen participates in osteogenesis as it does in mammalian bone. Our findings strongly suggest that regenerating scales can be used as a model of osteogenesis in vertebrates.     

Nodal cilia dynamics and the specification of the left/right axis in early vertebrate embryo development

Nodal cilia dynamics is a key factor for left/right axis determination in mouse embryos through the induction of a leftward fluid flow. So far it has not been clearly established how such dynamics is able to induce the asymmetric leftward flow within the node. Herein we propose that an asymmetric two-phase nonplanar beating cilia dynamics that involves the bending of the ciliar axoneme is responsible for the leftward fluid flow. We support our proposal with a host of hydrodynamic arguments, in silico experiments and in vivo video microscopy data in wild-type embryos and inv mutants. Our phenomenological modeling approach underscores how the asymmetry and speed of the flow depends on different relevant parameters. In addition, we discuss how the combination of internal and external mechanisms might cause the two-phase beating cilia dynamics.