Induction of preputium eversion by peptides, serotonin receptor antagonists, and selective serotonin reuptake inhibitors in Biomphalaria glabrata

Abstract. Eversion of the preputium is one of the initial steps in the male copulatory behavior of freshwater pulmonates. Previous experiments have shown that serotonergic mechanisms are involved in eversion in the snail Biomphalaria glabrata because the vertebrate 5-HT₁ receptor antagonist methiothepin caused long-lasting eversion in a dose-dependent manner. In this study, we tested a variety of serotonergic receptor ligands, bioactive peptides, and selective serotonin reuptake inhibitors (SSRIs) for their ability to induce preputium eversion in B . glabrata in order to elucidate the physiological mechanism of eversion. Of 15 compounds tested, five significantly induced preputium eversion: the serotonin receptor antagonists methiothepin (1 and 10 μM; p<0.0001), cyproheptadine (1–10 μM; p<0.007–0.0001), and mianserin (5–50 μM; p<0.01–0.001), the molluscan cardioactive peptide FMRFamide (10 and 50 μM; p<0.0002–0.0001), and the SSRI fluoxetine (=Prozac, 10–100 μM; p<0.0003–0.0001). Serotonin itself neither induced eversion nor blocked methiothepin-induced eversion. This suggests that fluoxetine is not acting as an SSRI, but potentially as a receptor ligand. These preliminary data shed light on the possible physiological mechanism of preputium eversion in B . glabrata and suggest similarity with that of the model freshwater gastropod Lymnaea stagnalis .     

Microtubule pivoting enables mitotic spindle assembly in S. cerevisiae

To assemble a bipolar spindle, microtubules emanating from two poles must bundle into an antiparallel midzone, where plus end–directed motors generate outward pushing forces to drive pole separation. Midzone cross-linkers and motors display only modest preferences for antiparallel filaments, and duplicated poles are initially tethered together, an arrangement that instead favors parallel interactions. Pivoting of microtubules around spindle poles might help overcome this geometric bias, but the intrinsic pivoting flexibility of the microtubule–pole interface has not been directly measured, nor has its importance during early spindle assembly been tested. By measuring the pivoting of microtubules around isolated yeast spindle poles, we show that pivoting flexibility can be modified by mutating a microtubule-anchoring pole component, Spc110. By engineering mutants with different flexibilities, we establish the importance of pivoting in vivo for timely pole separation. Our results suggest that passive thermal pivoting can bring microtubules from side-by-side poles into initial contact, but active minus end–directed force generation will be needed to achieve antiparallel alignment.     

Nuclear Envelope Protein Lem2 is Required for Mouse Development and Regulates MAP and AKT Kinases

The nuclear lamina, along with associated nuclear membrane proteins, is a nexus for regulating signaling in the nucleus. Numerous human diseases arise from mutations in lamina proteins, and experimental models for these disorders have revealed aberrant regulation of various signaling pathways. Previously, we reported that the inner nuclear membrane protein Lem2, which is expressed at high levels in muscle, promotes the differentiation of cultured myoblasts by attenuating ERK signaling. Here, we have analyzed mice harboring a disrupted allele for the Lem2 gene (Lemd2). No gross phenotypic defects were seen in heterozygotes, although muscle regeneration induced by cardiotoxin was delayed. By contrast, homozygous Lemd2 knockout mice died by E11.5. Although many normal morphogenetic hallmarks were observed in E10.5 knockout embryos, most tissues were substantially reduced in size. This was accompanied by activation of multiple MAP kinases (ERK1/2, JNK, p38) and AKT. Knockdown of Lem2 expression in C2C12 myoblasts also led to activation of MAP kinases and AKT. These findings indicate that Lemd2 plays an essential role in mouse embryonic development and that it is involved in regulating several signaling pathways. Since increased MAP kinase and AKT/mTORC signaling is found in other animal models for diseases linked to nuclear lamina proteins, LEMD2 should be considered to be another candidate gene for human disease.     

GRAVITROPISM IN ABSCISIC-ACID DEFICIENT SEEDLINGS OF ZEA MAYS

We did not detect any abscisic acid (ABA) in roots or leaves of carotenoid-deficient mutants of Zea mays. Similarly, we did not detect any ABA in roots or leaves of seedlings treated with Fluridone (an inhibitor of carotenogenesis) even after subjecting them to polyethylene glycol (PEG)-induced moisture stress. Primary roots of untreated, Fluridone-treated, and mutant seedlings were strongly graviresponsive. These results suggest that 1) ABA is not necessary for positive gravitropism by primary roots of these cultivars of Z. mays, and 2) ABA is synthesized via the carotenoid pathway.     

Mitochondrial Changes in Ageing Caenorhabditis elegans – What Do We Learn from Superoxide Dismutase Knockouts?

One of the most popular damage accumulation theories of ageing is the mitochondrial free radical theory of ageing (mFRTA). The mFRTA proposes that ageing is due to the accumulation of unrepaired oxidative damage, in particular damage to mitochondrial DNA (mtDNA). Within the mFRTA, the "vicious cycle" theory further proposes that reactive oxygen species (ROS) promote mtDNA mutations, which then lead to a further increase in ROS production. Recently, data have been published on Caenorhabditis elegans mutants deficient in one or both forms of mitochondrial superoxide dismutase (SOD). Surprisingly, even double mutants, lacking both mitochondrial forms of SOD, show no reduction in lifespan. This has been interpreted as evidence against the mFRTA because it is assumed that these mutants suffer from significantly elevated oxidative damage to their mitochondria. Here, using a novel mtDNA damage assay in conjunction with related, well established damage and metabolic markers, we first investigate the age-dependent mitochondrial decline in a cohort of ageing wild-type nematodes, in particular testing the plausibility of the "vicious cycle" theory. We then apply the methods and insights gained from this investigation to a mutant strain for C. elegans that lacks both forms of mitochondrial SOD. While we show a clear age-dependent, linear increase in oxidative damage in WT nematodes, we find no evidence for autocatalytic damage amplification as proposed by the "vicious cycle" theory. Comparing the SOD mutants with wild-type animals, we further show that oxidative damage levels in the mtDNA of SOD mutants are not significantly different from those in wild-type animals, i.e. even the total loss of mitochondrial SOD did not significantly increase oxidative damage to mtDNA. Possible reasons for this unexpected result and some implications for the mFRTA are discussed.     

Protocols for the assurance of microarray data quality and process control

Microarrays represent a powerful technology that provides the ability to simultaneously measure the expression of thousands of genes. However, it is a multi-step process with numerous potential sources of variation that can compromise data analysis and interpretation if left uncontrolled, necessitating the development of quality control protocols to ensure assay consistency and high-quality data. In response to emerging standards, such as the minimum information about a microarray experiment standard, tools are required to ascertain the quality and reproducibility of results within and across studies. To this end, an intralaboratory quality control protocol for two color, spotted microarrays was developed using cDNA microarrays from in vivo and in vitro dose-response and time-course studies. The protocol combines: (i) diagnostic plots monitoring the degree of feature saturation, global feature and background intensities, and feature misalignments with (ii) plots monitoring the intensity distributions within arrays with (iii) a support vector machine (SVM) model. The protocol is applicable to any laboratory with sufficient datasets to establish historical high- and low-quality data.     

Molecular mechanisms of survival and apoptosis in RAW 264.7 macrophages under oxidative stress

Organisms living in an aerobic environment are continuously exposed to reactive oxygen species (ROS). Apoptosis of cells can be induced by ROS and cells also develop negative feedback mechanisms to limit ROS induced cell death. In this study, RAW264.7 murine macrophage cells were treated with H₂O₂ and cDNA microarray technique was used to produce gene expression profiles. We found that H₂O₂ treatment caused up-regulation of stress, survival and apoptosis related genes, and down-regulation of growth and cell cycle promoting genes. Numerous genes of metabolism pathways showed special expression patterns under oxidative stress: glycolysis and lipid synthesis related genes were down-regulated whereas the genes of lipid catabolism and protein synthesis were up-regulated. We also identified several signaling molecules as ROS-responsive, including p53, Akt, NF- B, ERK, JNK, p38, PKC and INF- . They played important roles in the process of apoptosis or cell survival. Finally, an interactive pathway involved in cellular response to oxidative stress was proposed to provide some insight into the molecular events of apoptosis induced by ROS and the feedback mechanisms involved in cell survival.Y. Zhang and C.C. Fong contributed equally to this work.