Mediator subunits MED1 and MED24 cooperatively contribute to pubertal mammary gland development and growth of breast carcinoma cells

The Mediator subunit MED1 is essential for mammary gland development and lactation, whose contribution through direct interaction with estrogen receptors (ERs) is restricted to involvement in pubertal mammary gland development and luminal cell differentiation. Here, we provide evidence that the MED24-containing submodule of Mediator functionally communicates specifically with MED1 in pubertal mammary gland development. Mammary glands from MED1/MED24 double heterozygous knockout mice showed profound retardation in ductal branching during puberty, while single haploinsufficient glands developed normally. DNA synthesis of both luminal and basal cells were impaired in double mutant mice, and the expression of ER-targeted genes encoding E2F1 and cyclin D1, which promote progression through the G(1)/S phase of the cell cycle, was attenuated. Luciferase reporter assays employing double mutant mouse embryonic fibroblasts showed selective impairment in ER functions. Various breast carcinoma cell lines expressed abundant amounts of MED1, MED24, and MED30, and attenuated expression of MED1 and MED24 in breast carcinoma cells led to attenuated DNA synthesis and growth. These results indicate functional communications between the MED1 subunit and the MED24-containing submodule that mediate estrogen receptor functions and growth of both normal mammary epithelial cells and breast carcinoma cells.     

Global analysis of hematopoietic and vascular endothelial gene expression by tissue specific microarray profiling in zebrafish

In this study, we utilize fluorescent activated cell sorting (FACS) of cells from transgenic zebrafish coupled with microarray analysis to globally analyze expression of cell type specific genes. We find that it is possible to isolate cell populations from Tg(fli1:egfp)(y1) zebrafish embryos that are enriched in vascular, hematopoietic and pharyngeal arch cell types. Microarray analysis of GFP+ versus GFP- cells isolated from Tg(fli1:egfp)(y1) embryos identifies genes expressed in hematopoietic, vascular and pharyngeal arch tissue, consistent with the expression of the fli1:egfp transgene in these cell types. Comparison of expression profiles from GFP+ cells isolated from embryos at two different time points reveals that genes expressed in different fli1+ cell types display distinct temporal expression profiles. We also demonstrate the utility of this approach for gene discovery by identifying numerous previously uncharacterized genes that we find are expressed in fli1:egfp-positive cells, including new markers of blood, endothelial and pharyngeal arch cell types. In parallel, we have developed a database to allow easy access to both our microarray and in situ results. Our results demonstrate that this is a robust approach for identification of cell type specific genes as well as for global analysis of cell type specific gene expression in zebrafish embryos.     

Chloroplast DNA variation and geographical structure of the Aristolochia kaempferi group (Aristolochiaceae)

The present study documents cpDNA variation in the Aristolochia kaempferi group (Aristolochiaceae), which consists of one Chinese and all Japanese and Taiwanese species of the subgenus Siphisia. In a phylogenetic analysis based on the nucleotide sequences of the matK gene, and the atpB-rbcL and trnS-trnG intergenic spacer regions, 38 haplotypes were recognized in the A. kaempferi group and as many as 24 within A. kaempferi. This is the most haplotypes reported for a single species to date. Although six highly significant major clades were identified in the phylogenetic analysis, they were not congruent with previous classifications. This might be attributed to the specific speciation process, such as convergent evolution, incomplete lineage sorting, and/or reticulate evolution. The six major clades had a clear geographical distribution pattern and were significantly associated with geographical distribution of haplotypes in a nested clade analysis and AMOVA. The results allow us to deduce a scenario in which multiple contractions and expansions of the geographical ranges brought about by Quaternary climatic oscillations affected the patterns of genetic diversity. The present geographic patterns of haplotype distribution within the A. kaempferi group can be explained by the last postglacial range expansion from different refugia, and the boundaries may be suture zones.     

ANGLE: a sequencing errors resistant program for predicting protein coding regions in unfinished cDNA

In the process of making full-length cDNA, predicting protein coding regions helps both in the preliminary analysis of genes and in any succeeding process. However, unfinished cDNA contains artifacts including many sequencing errors, which hinder the correct evaluation of coding sequences. Especially, predictions of short sequences are difficult because they provide little information for evaluating coding potential. In this paper, we describe ANGLE, a new program for predicting coding sequences in low quality cDNA. To achieve error-tolerant prediction, ANGLE uses a machine-learning approach, which makes better expression of coding sequence maximizing the use of limited information from input sequences. Our method utilizes not only codon usage, but also protein structure information which is difficult to be used for stochastic model-based algorithms, and optimizes limited information from a short segment when deciding coding potential, with the result that predictive accuracy does not depend on the length of an input sequence. The performance of ANGLE is compared with ESTSCAN on four dataset each of them having a different error rate (one frame-shift error or one substitution error per 200-500 nucleotides) and on one dataset which has no error. ANGLE outperforms ESTSCAN by 9.26% in average Matthews's correlation coefficient on short sequence dataset (< 1000 bases). On long sequence dataset, ANGLE achieves comparable performance.     

MCP-1 expressed by osteoclasts stimulates osteoclastogenesis in an autocrine/paracrine manner

Monocyte chemoattractant protein-1 (MCP-1) is a chemokine that plays a critical role in the recruitment and activation of leukocytes. Here, we describe that multinuclear osteoclast formation was significantly inhibited in cells derived from MCP-1-deficient mice. MCP-1 has been implicated in the regulation of osteoclast cell-cell fusion; however defects of multinuclear osteoclast formation in the cells from mice deficient in DC-STAMP, a seven transmembrane receptor essential for osteoclast cell-cell fusion, was not rescued by recombinant MCP-1. The lack of MCP-1 in osteoclasts resulted in a down-regulation of DC-STAMP, NFATc1, and cathepsin K, all of which were highly expressed in normal osteoclasts, suggesting that osteoclast differentiation was inhibited in MCP-1-deficient cells. MCP-1 alone did not induce osteoclastogenesis, however, the inhibition of osteoclastogenesis in MCP-1-deficient cells was restored by addition of recombinant MCP-1, indicating that osteoclastogenesis was regulated in an autocrine/paracrine manner by MCP-1 under the stimulation of RANKL in osteoclasts.     

Methamphetamine directly accelerates beating rate in cardiomyocytes by increasing Ca entry via L-type Ca channel

Methamphetamine induces several cardiac dysfunctions, which leads to arrhythmia, cardiac failure and sudden cardiac death. Although these cardiac alterations elicited by methamphetamine were thought to be due to an indirect action of methamphetamine, namely, an excessive catecholamine release from synaptic terminals, while it seems likely that methamphetamine directly modulates the functioning of cardiomyocytes independent of neurotransmitters. However, the direct effects of methamphetamine on cardiomyocytes are still not clear. We show that methamphetamine directly accelerates the beating rate and alters Ca²⁺ oscillation pattern in cultured neonatal rat cardiomyocytes. Adrenergic receptor antagonists did not block the methamphetamine-induced alterations in cardiomyocytes. Treatment with a ryanodine receptor type 2 inhibitor and a sarcoplasmic reticulum Ca²⁺-ATPase inhibitor did not affect these responses, either. In contrast, the L-type Ca²⁺ channel inhibitor nifedipine eradicated these responses. Furthermore, methamphetamine elevated the internal free Ca²⁺ concentration in HEK-293T cells stably transfected with the L-type Ca²⁺ channel α1C subunit. In neonatal rat cardiomyocytes, methamphetamine accelerates beating rate and alters Ca²⁺ oscillation pattern by increasing Ca²⁺ entry via the L-type Ca²⁺ channels independent of any neurotransmitters.     

Resuscitation-promoting factors as lytic enzymes for bacterial growth and signaling

Resuscitation-promoting factor (Rpf) is a muralytic enzyme that increases the culturability of dormant bacteria. Recently, considerable progress has been made in understanding the structure, function and physiological role of Rpfs in different organisms, most notably the major human pathogen, Mycobacterium tuberculosis , which encodes multiple rpf -like genes. A key unresolved question, however, concerns the relationship between the predicted biochemical activity of Rpfs – cleavage of the β-1,4 glycosidic bond in the glycan backbone of peptidoglycan – and their effect on culturability. In M. tuberculosis , the interaction between RpfB and the d,l -endopeptidase, Rpf interacting protein A (RipA), enables these proteins to synergistically degrade peptidoglycan to facilitate growth. Furthermore, the combined action of Rpfs with RipA and other peptidoglycan hydrolases might produce muropeptides that could exert diverse biological effects through host and/or bacterial signaling, the latter involving serine/threonine protein kinases. Here, we explore these possibilities in the context of the structure and composition of mycobacterial peptidoglycan. Clearly, a deeper understanding of the role of Rpfs and associated peptidoglycan remodeling enzymes in bacterial growth and culturability is necessary to establish the significance of dormancy and resuscitation in diseases such as tuberculosis, which are associated with long-term persistence of viable bacterial populations recalcitrant to antibiotic and immune clearance.     

Changes in Extracellular Kynurenic Acid Concentrations in Rat Prefrontal Cortex After <Emphasis Type="SmallCaps">d</Emphasis>-Kynurenine Infusion: An In vivo Microdialysis Study

Using a microdialysis technique, we continuously infused d-kynurenine (KYN) (0, 50, and 100 μM) into the prefrontal cortices (PFCs) of male Sprague–Dawley rats. We then used column-switching high-performance liquid chromatography to assess the alterations in the concentration of kynurenic acid (KYNA)—an antagonist of N-methyl-d-aspartate and α7 nicotinic acetylcholine receptors—in the extracellular fluid in the PFC. Local infusion of d-KYN into the PFC remarkably increased the extracellular KYNA concentration, indicating that d-KYN is metabolized to KYNA in the PFC. The d-KYN-induced increase in KYNA levels was significantly attenuated by the co-administration of 3-methylpyrazole-5-carboxylic acid (AS057278)—a specific inhibitor of d-amino acid oxidase (DAAO). These results suggest that DAAO may be involved in the production of KYNA from d-KYN in the PFC in vivo.     

Female exhibited severe cognitive impairment in type 2 diabetes mellitus mice

AimsSex-specific medicine has been highlighted as a different approach to the diagnosis and treatment of diseases between men and women. Type 2 diabetes has been reported to be a risk factor for cognitive impairment. Here, we investigated the sex difference in cognitive function associated with diabetes using KKAy mice.Main methodsCognitive function was evaluated by shuttle avoidance test and Morris water maze test. Changes in gene expression in the brain were evaluated by PCR array and confirmed by quantitative RT-PCR. To evaluate the effect of estradiol, some female KKAy were ovariectomized and treated with or without estradiol.Key findingsIn KKAy mice, female significantly exhibited impaired cognitive function compared with male, while there was no sex difference in these cognitive functions in C57BL6, wild-type mice. Female KKAy mice showed hyperinsulinemia, impaired glucose tolerance and increased oxidative stress compared with male KKAy mice. Female KKAy also showed a significant decrease in peroxisome proliferators-activated receptor (PPAR)-γ expression in the brain compared with male KKAy. Estradiol treatment improved the insulin resistance and higher superoxide production, but failed to improve the cognitive task performance, serum insulin level and lower expression of PPAR-γ.SignificanceIn diabetic mice, female showed significantly impaired cognitive function, with greater insulin resistance, lower expression of PPAR-γ and higher superoxide production compared with male. Estrogen had little effect on cognitive function. These results indicate that a sex-specific approach to cognitive impairment is necessary for diabetic patients, especially for women.