Aprotinin Attenuates the Elevation of Pulmonary Vascular Resistance After Cardiopulmonary Bypass

Pulmonary vascular resistance (PVR) is generally believed to be elevated after cardiopulmonary bypass (CPB) due to whole body inflammation. Aprotinin has an anti-inflammatory action, and it was hypothesized that aprotinin would attenuate the PVR increase induced by CPB. Ten mongrel dogs were placed under moderately hypothermic CPB for 2 hr. The experimental animals were divided into a control group (n=5, group I) and an aprotinin group (n=5, group II). In group II, aprotinin was administered during pre-bypass (50,000 KIU/kg) and post-bypass (10,000 KIU/kg) periods. Additional aprotinin (50,000 KIU/kg) was mixed in CPB priming solution. PVRs at pre-bypass and post-bypass 0, 1, 2, 3 hr were calculated, and lung tissue was obtained after the experiment. Post-bypass PVRs were significantly higher than prebypass levels in all animals (n=10, p<0.001). PVR elevation in group II was less than in group I at 3 hr post-bypass (p=0.0047). Water content of the lung was lower in group II (74±9.4%) compared to that of group I (83±9.5%), but the difference did not reach significance (p=0.076). Pathological examination showed a near normal lung structure in group II, whereas various inflammatory reactions were observed in group I. We concluded that aprotinin may attenuate CPB-induced PVR elevation through its anti-inflammatory effect

Nutrient-Regulated Antisense and Intragenic RNAs Modulate a Signal Transduction Pathway in Yeast

The budding yeast Saccharomyces cerevisiae alters its gene expression profile in response to a change in nutrient availability. The PHO system is a well-studied case in the transcriptional regulation responding to nutritional changes in which a set of genes (PHO genes) is expressed to activate inorganic phosphate (Pi) metabolism for adaptation to Pi starvation. Pi starvation triggers an inhibition of Pho85 kinase, leading to migration of unphosphorylated Pho4 transcriptional activator into the nucleus and enabling expression of PHO genes. When Pi is sufficient, the Pho85 kinase phosphorylates Pho4, thereby excluding it from the nucleus and resulting in repression (i.e., lack of transcription) of PHO genes. The Pho85 kinase has a role in various cellular functions other than regulation of the PHO system in that Pho85 monitors whether environmental conditions are adequate for cell growth and represses inadequate (untimely) responses in these cellular processes. In contrast, Pho4 appears to activate some genes involved in stress response and is required for G1 arrest caused by DNA damage. These facts suggest the antagonistic function of these two players on a more general scale when yeast cells must cope with stress conditions. To explore general involvement of Pho4 in stress response, we tried to identify Pho4-dependent genes by a genome-wide mapping of Pho4 and Rpo21 binding (Rpo21 being the largest subunit of RNA polymerase II) using a yeast tiling array. In the course of this study, we found Pi- and Pho4-regulated intragenic and antisense RNAs that could modulate the Pi signal transduction pathway. Low-Pi signal is transmitted via certain inositol polyphosphate (IP) species (IP7) that are synthesized by Vip1 IP6 kinase. We have shown that Pho4 activates the transcription of antisense and intragenic RNAs in the KCS1 locus to down-regulate the Kcs1 activity, another IP6 kinase, by producing truncated Kcs1 protein via hybrid formation with the KCS1 mRNA and translation of the intragenic RNA, thereby enabling Vip1 to utilize more IP6 to synthesize IP7 functioning in low-Pi signaling. Because Kcs1 also can phosphorylate these IP7 species to synthesize IP8, reduction in Kcs1 activity can ensure accumulation of the IP7 species, leading to further stimulation of low-Pi signaling (i.e., forming a positive feedback loop). We also report that genes apparently not involved in the PHO system are regulated by Pho4 either dependent upon or independent of the Pi conditions, and many of the latter genes are involved in stress response. In S. cerevisiae, a large-scale cDNA analysis and mapping of RNA polymerase II binding using a high-resolution tiling array have identified a large number of antisense RNA species whose functions are yet to be clarified. Here we have shown that nutrient-regulated antisense and intragenic RNAs as well as direct regulation of structural gene transcription function in the response to nutrient availability. Our findings also imply that Pho4 is present in the nucleus even under high-Pi conditions to activate or repress transcription, which challenges our current understanding of Pho4 regulation

Transcriptional Repression by the Pho4 Transcription Factor Controls the Timing of SNZ1 Expression▿ †

Nutrient-sensing kinases play important roles for the yeast Saccharomyces cerevisiae to adapt to new nutrient conditions when the nutrient status changes. Our previous global gene expression analysis revealed that the Pho85 kinase, one of the yeast nutrient-sensing kinases, is involved in the changes in gene expression profiles when yeast cells undergo a diauxic shift. We also found that the stationary phase-specific genes SNZ1 and SNO1, whch share a common promoter, are not properly induced when Pho85 is absent. To examine the role of the kinase in SNZ1/SNO1 regulation, we analyzed their expression during the growth of various yeast mutants, including those affecting Pho85 function or lacking the Pho4 transcription factor, an in vivo substrate of Pho85, and tested Pho4 binding by chromatin immunoprecipitation. Pho4 exhibits temporal binding to the SNZ1/SNO1 promoter to down-regulate the promoter activity, and a Δpho4 mutation advances the timing of SNZ1/SNO1 expression. SNZ2, another member of the SNZ/SNO family, is expressed at an earlier growth stage than SNZ1, and Pho4 does not affect this timing, although Pho85 is required for SNZ2 expression. Thus, Pho4 appears to regulate the different timing of the expression of the SNZ/SNO family members. Pho4 binding to the SNZ1/SNO1 promoter is accompanied by alterations in chromatin structure, and Rpd3 histone deacetylase is required for the proper timing of SNZ1/SNO1 expression, while Asf1 histone chaperone is indispensable for their expression. These results imply that Pho4 plays positive and negative roles in transcriptional regulation, with both cases involving structural changes in its target chromatin

Prdm8 Regulates the Morphological Transition at Multipolar Phase during Neocortical Development

<div><p>Here, we found that the PR domain protein Prdm8 serves as a key regulator of the length of the multipolar phase by controlling the timing of morphological transition. We used a mouse line with expression of <i>Prdm8</i>-mVenus reporter and found that Prdm8 is predominantly expressed in the middle and upper intermediate zone during both the late and terminal multipolar phases. Prdm8 expression was almost coincident with Unc5D expression, a marker for the late multipolar phase, although the expression of Unc5D was found to be gradually down-regulated to the point at which mVenus expression was gradually up-regulated. This expression pattern suggests the possible involvement of Prdm8 in the control of the late and terminal multipolar phases, which controls the timing for morphological transition. To test this hypothesis, we performed gain- and loss-of-function analysis of neocortical development by using in utero electroporation. We found that the knockdown of Prdm8 results in premature change from multipolar to bipolar morphology, whereas the overexpression of Prdm8 maintained the multipolar morphology. Additionally, the postnatal analysis showed that the Prdm8 knockdown stimulated the number of early born neurons, and differentiated neurons located more deeply in the neocortex, however, majority of those cells could not acquire molecular features consistent with laminar location. Furthermore, we found the candidate genes that were predominantly utilized in both the late and terminal multipolar phases, and these candidate genes included those encoding for guidance molecules. In addition, we also found that the expression level of these guidance molecules was inhibited by the introduction of the Prdm8 expression vector. These results indicate that the Prdm8-mediated regulation of morphological changes that normally occur during the late and terminal multipolar phases plays an important role in neocortical development.</p></div

Microarray expression analyses of mVenus-positive versus mVenus-negative cells from E15.5 <i>Prdm8</i>-mVenus mouse neocortex.

<p>The genes with expression levels more than 2.4 fold higher in <i>Prdm8</i>-mVenus positive cells compared to mVenus negative cells are listed. From this analysis (n = 2), we have identified several genes showing specific expression within middle-IZ and/or upper-IZ.</p

IZ or VZ/SVZ-expressed genes showing changes in microarray analyses from E15.5 <i>Prdm8</i>-mVenus mouse neocortex.

<p>IZ or VZ/SVZ-expressed genes showing changes in microarray analyses from E15.5 <i>Prdm8</i>-mVenus mouse neocortex.</p

Prdm8 alters layer formation in the neocortex.

<p>In utero electroporation of any one of control (pCAG-IRES-EGFP with pCAG-IRES-Puro; A), or Prdm8 gain-of-function (pCAG-IRES-EGFP with pCAG-Prdm8; B), or Prdm8 loss-of-function (pCAG-IRES-EGFP with pPrdm8sh#629; C) vectors were carried out at E12.5, and the brains were analyzed at P5. The cortex was divided into 10 bins and the percentage of EGFP-positive cells were quantified (D). The distribution of EGFP-positive cells is significantly increased in the upper bins (bins8–10) and reduced in lower bins (bin5) in the case of the pCAG-Prdm8-electroporated brains, and significantly decreased in upper bins (bins4–6), and increased in lower bins (bins1, 2) in the pPrdm8sh-electroporated brains. The number of counted cells was about 137 cells. Data represents the mean ± SD (n = 6 slices from 3 individuals); *p<0.05, **p<0.01. High-power images showing that the molecular features of control, Prdm8 gain-of-function, or loss-of-function cells in the neocortex, stained with Tbr1 (E, F, G), Ctip2 (K, L, M), RORb (K, L, M) and Brn2 (H, I, J). The percentage of each layer marker-positive EGFP-positive cells located in each layer position was quantified (N). The number of counted cells was about 184 cells. Data represents the mean ± SD (n>4 slices from 4 individuals); **p<0.01. Scale bars: 100 µm (A,E).</p

Candidate genes, preferentially expressed in the late-MP and/or terminal-MP phases.

<p>Ten candidate genes were selected by the validation of DNA microarray data (A) (n = 2). Quantitative real-time PCR data shows the expression level of some candidate genes was suppressed by the introduction of pCAG-Prdm8 (B) (n = 4) or pCAG-Unc5D (C) (n = 3). Schematic drawing of a working hypothesis of this study (D).</p

Prdm8 expression is mainly restricted to the middle- and upper -IZ during neocortical development.

<p>Immunostaining reveals that Prdm8 is weakly expressed in the lower-IZ (A, B). Prdm8 is highly expressed in the middle-IZ and upper-IZ, where its expression almost overlaps with Unc5d expression (B′, B″), which is a marker for late-MP phase, at E15.5. Prdm8-positive cells do not overlap with the Tbr2-positive cells (C), and some Prdm8-positive cells in the lower-IZ are positive for NeuroD1 (D). The mVenus expression pattern in transgenic mouse (E) is similar to the immunostaining pattern obtained with anti-Prdm8 antibody at E15.5 (See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086356#pone.0086356.s001" target="_blank">Figure S1A–D</a>, D′). The upregulation of Prdm8 (indicated by the asterisk) occurs in the MP cells at the late-MP phase (F, G, G′, G″). For improved visualization of the cell morphology during the MP phase, the <i>Prdm8</i>-mVenus embryo was electroporated at E12.5 with the CAG-mCherry vector, and analyzed 48 h later. Depending on their position within the IZ, the mCherry-positive MP or BP cells (white or yellow arrows; MP cells in the upper-IZ or lower-IZ, respectively. White arrowheads; BP cells in the upper-IZ) exhibit distinct levels of Prdm8 expression (H, H′, H″). Schematic drawing of the sequential expression of NeuroD1, Unc5D, and Prdm8 that coincides with the process of early post-mitotic differentiation in the neocortical development (K). The nuclei are stained with DAPI in A and E. Scale bars: 100 µm.</p