Differential Enhancement of a Cutaneous HPV Promoter by ΔNP63α, JUN and Mutant p53

The mechanism through which cutaneous papillomaviruses induce lesions is largelyunknown. Ectopic expression of the ΔNP63α isoform highly increased the viral promoteractivity. The co-expression of c-Jun mediated and increased the ΔNP63α activity by bindingto the AP-1 site in an enhancer region of the HPV 20 URR. This strong activation by ΔNP63αis diminished in the presence of wtp53 and abolished by the simultaneous expression of “hotspot”mutant p53 R248W. We demonstrate that c-Jun is responsible for the viral promoteractivation through its direct interaction with both ΔNP63α and wtp53. The down-regulationby p53 mutant R248W is accompanied by reduced protein levels of ΔNP63α andphosphorylated c-Jun. The data presented in this study provide insight into a possiblemechanism through which these cellular proteins may modulate a cutaneous papillomavirusgenome to induce viral replication, latent infection or malignant trasnformation.     

The DNA Damage Response and Checkpoint Adaptation in Saccharomyces cerevisiae: Distinct Roles for the Replication Protein A2 (Rfa2) N-Terminus

In response to DNA damage, two general but fundamental processes occur in the cell: (1) a DNA lesion is recognized and repaired, and (2) concomitantly, the cell halts the cell cycle to provide a window of opportunity for repair to occur. An essential factor for a proper DNA-damage response is the heterotrimeric protein complex Replication Protein A (RPA). Of particular interest is hyperphosphorylation of the 32-kDa subunit, called RPA2, on its serine/threonine-rich amino (N) terminus following DNA damage in human cells. The unstructured N-terminus is often referred to as the phosphorylation domain and is conserved among eukaryotic RPA2 subunits, including Rfa2 in Saccharomyces cerevisiae. An aspartic acid/alanine-scanning and genetic interaction approach was utilized to delineate the importance of this domain in budding yeast. It was determined that the Rfa2 N-terminus is important for a proper DNA-damage response in yeast, although its phosphorylation is not required. Subregions of the Rfa2 N-terminus important for the DNA-damage response were also identified. Finally, an Rfa2 N-terminal hyperphosphorylation-mimetic mutant behaves similarly to another Rfa1 mutant (rfa1-t11) with respect to genetic interactions, DNA-damage sensitivity, and checkpoint adaptation. Our data indicate that post-translational modification of the Rfa2 N-terminus is not required for cells to deal with “repairable” DNA damage; however, post-translational modification of this domain might influence whether cells proceed into M-phase in the continued presence of unrepaired DNA lesions as a “last-resort” mechanism for cell survival.     

Remodeling of the Rad51 DNA Strand-Exchange Protein by the Srs2 Helicase

Homologous recombination is associated with the dynamic assembly and disassembly of DNA–protein complexes. Assembly of a nucleoprotein filament comprising ssDNA and the RecA homolog, Rad51, is a key step required for homology search during recombination. The budding yeast Srs2 DNA translocase is known to dismantle Rad51 filament in vitro. However, there is limited evidence to support the dismantling activity of Srs2 in vivo. Here, we show that Srs2 indeed disrupts Rad51-containing complexes from chromosomes during meiosis. Overexpression of Srs2 during the meiotic prophase impairs meiotic recombination and removes Rad51 from meiotic chromosomes. This dismantling activity is specific for Rad51, as Srs2 Overexpression does not remove Dmc1 (a meiosis-specific Rad51 homolog), Rad52 (a Rad51 mediator), or replication protein A (RPA; a single-stranded DNA-binding protein). Rather, RPA replaces Rad51 under these conditions. A mutant Srs2 lacking helicase activity cannot remove Rad51 from meiotic chromosomes. Interestingly, the Rad51-binding domain of Srs2, which is critical for Rad51-dismantling activity in vitro, is not essential for this activity in vivo. Our results suggest that a precise level of Srs2, in the form of the Srs2 translocase, is required to appropriately regulate the Rad51 nucleoprotein filament dynamics during meiosis.     

Coordination of Recombination with Meiotic Progression in the Caenorhabditis elegans Germline by KIN-18, a TAO Kinase That Regulates the Timing of MPK-1 Signaling

Meiosis is a tightly regulated process requiring coordination of diverse events. A conserved ERK/MAPK-signaling cascade plays an essential role in the regulation of meiotic progression. The Thousand And One kinase (TAO) kinase is a MAPK kinase kinase, the meiotic role of which is unknown. We have analyzed the meiotic functions of KIN-18, the homolog of mammalian TAO kinases, in Caenorhabditis elegans. We found that KIN-18 is essential for normal meiotic progression; mutants exhibit accelerated meiotic recombination as detected both by analysis of recombination intermediates and by crossover outcome. In addition, ectopic germ-cell differentiation and enhanced levels of apoptosis were observed in kin-18 mutants. These defects correlate with ectopic activation of MPK-1 that includes premature, missing, and reoccurring MPK-1 activation. Late progression defects in kin-18 mutants are suppressed by inhibiting an upstream activator of MPK-1 signaling, KSR-2. However, the acceleration of recombination events observed in kin-18 mutants is largely MPK-1-independent. Our data suggest that KIN-18 coordinates meiotic progression by modulating the timing of MPK-1 activation and the progression of recombination events. The regulation of the timing of MPK-1 activation ensures the proper timing of apoptosis and is required for the formation of functional oocytes. Meiosis is a conserved process; thus, revealing that KIN-18 is a novel regulator of meiotic progression in C. elegans would help to elucidate TAO kinase’s role in germline development in higher eukaryotes.     

Resection Activity of the Sgs1 Helicase Alters the Affinity of DNA Ends for Homologous Recombination Proteins in Saccharomyces cerevisiae

The RecQ helicase family is critical during DNA damage repair, and mutations in these proteins are associated with Bloom, Werner, or Rothmund-Thompson syndromes in humans, leading to cancer predisposition and/or premature aging. In the budding yeast Saccharomyces cerevisiae, mutations in the RecQ homolog, SGS1, phenocopy many of the defects observed in the human syndromes. One challenge to studying RecQ helicases is that their disruption leads to a pleiotropic phenotype. Using yeast, we show that the separation-of-function allele of SGS1, sgs1-D664Δ, has impaired activity at DNA ends, resulting in a resection processivity defect. Compromising Sgs1 resection function in the absence of the Sae2 nuclease causes slow growth, which is alleviated by making the DNA ends accessible to Exo1 nuclease. Furthermore, fluorescent microscopy studies reveal that, when Sgs1 resection activity is compromised in sae2Δ cells, Mre11 repair foci persist. We suggest a model where the role of Sgs1 in end resection along with Sae2 is important for removing Mre11 from DNA ends during repair.     

New Regulators of Clathrin-Mediated Endocytosis Identified in Saccharomyces cerevisiae by Systematic Quantitative Fluorescence Microscopy

Despite the importance of clathrin-mediated endocytosis (CME) for cell biology, it is unclear if all components of the machinery have been discovered and many regulatory aspects remain poorly understood. Here, using Saccharomyces cerevisiae and a fluorescence microscopy screening approach we identify previously unknown regulatory factors of the endocytic machinery. We further studied the top scoring protein identified in the screen, Ubx3, a member of the conserved ubiquitin regulatory X (UBX) protein family. In vivo and in vitro approaches demonstrate that Ubx3 is a new coat component. Ubx3-GFP has typical endocytic coat protein dynamics with a patch lifetime of 45 ± 3 sec. Ubx3 contains a W-box that mediates physical interaction with clathrin and Ubx3-GFP patch lifetime depends on clathrin. Deletion of the UBX3 gene caused defects in the uptake of Lucifer Yellow and the methionine transporter Mup1 demonstrating that Ubx3 is needed for efficient endocytosis. Further, the UBX domain is required both for localization and function of Ubx3 at endocytic sites. Mechanistically, Ubx3 regulates dynamics and patch lifetime of the early arriving protein Ede1 but not later arriving coat proteins or actin assembly. Conversely, Ede1 regulates the patch lifetime of Ubx3. Ubx3 likely regulates CME via the AAA-ATPase Cdc48, a ubiquitin-editing complex. Our results uncovered new components of the CME machinery that regulate this fundamental process.     

Fertility and Polarized Cell Growth Depends on eIF5A for Translation of Polyproline-Rich Formins in Saccharomyces cerevisiae

eIF5A is an essential and evolutionary conserved translation elongation factor, which has recently been proposed to be required for the translation of proteins with consecutive prolines. The binding of eIF5A to ribosomes occurs upon its activation by hypusination, a modification that requires spermidine, an essential factor for mammalian fertility that also promotes yeast mating. We show that in response to pheromone, hypusinated eIF5A is required for shmoo formation, localization of polarisome components, induction of cell fusion proteins, and actin assembly in yeast. We also show that eIF5A is required for the translation of Bni1, a proline-rich formin involved in polarized growth during shmoo formation. Our data indicate that translation of the polyproline motifs in Bni1 is eIF5A dependent and this translation dependency is lost upon deletion of the polyprolines. Moreover, an exogenous increase in Bni1 protein levels partially restores the defect in shmoo formation seen in eIF5A mutants. Overall, our results identify eIF5A as a novel and essential regulator of yeast mating through formin translation. Since eIF5A and polyproline formins are conserved across species, our results also suggest that eIF5A-dependent translation of formins could regulate polarized growth in such processes as fertility and cancer in higher eukaryotes.     

Genome sequence of the free-living aerobic spirochete Turneriella parva type strain (HT), and emendation of the species Turneriella parva

Turneriella parva Levett et al. 2005 is the only species of the genus Turneriella which was established as a result of the reclassification of Leptospira parva Hovind-Hougen et al. 1982. Together with Leptonema and Leptospira, Turneriella constitutes the family Leptospiraceae, within the order Spirochaetales. Here we describe the features of this free-living aerobic spirochete together with the complete genome sequence and annotation. This is the first complete genome sequence of a member of the genus Turneriella and the 13^th member of the family Leptospiraceae for which a complete or draft genome sequence is now available. The 4,409,302 bp long genome with its 4,169 protein-coding and 45 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.     

Oxysterols As Indices of Oxidative Stress in Man After Paraquat Ingestion

The aim of this study is to evaluate oxidative stress in man after paraquat ingestion by analyzing 7 &#102 - and 7 &#103 -hydroperoxycholest-5-en-3 &#103 -ol (7 &#102 - and 7 &#103 -OOH) as well as oxysterols, cholesterol oxidation products, as indices of lipid peroxidation. Lung, kidney, and liver were collected at autopsy from seven patients with paraquat poisoning and seven controls matched for age and sex. We identified for the first time 7-ketocholesterol (7-keto) and 7-hydroxycholesterol (7 &#102 -OH and 7 &#103 -OH) in human kidney by LC-MS. Next, we quantified 7 &#102 -OOH and 7 &#103 -OOH by HPLC with postcolumn chemiluminescence as well as oxysterols by HPLC-UV. Both 7 &#102 -OOH and 7 &#103 -OOH detected in lung and kidney from the controls were as low as the paraquat group. In contrast, we found both 7-keto and 7 &#103 -OH in lung and 7-keto in kidney from the paraquat group were significantly higher than from the controls. This is the first report on accumulated oxysterols in lung and kidney from human paraquat poisoning. It seems to reflect greater oxidative stress in the pathology of paraquat intoxication.     

ΔNp63α Levels Correlate with Clinical Tumor Response to Cisplatin

After exposure to damaging agents, the p53 tumor suppressor is stabilized mediating cell cycle arrest and apoptosis. p53 family member, ΔNp63α promotes cell proliferation and accelerates tumor growth. We previously found that the genotoxic stress agents induced a decrease of ΔNp63α . We further observed that genotoxic stress mediated phosphorylation of ΔNp63α targeting it into proteasome degradation. Here, we found that high ΔNp63 protein levels in primary tumors accurately predicted response to platinum based chemotherapy and a favorable outcome in head and neck cancer patients. Our data suggest that degradation of ΔNp63α is part of the cellular response to DNA damage in head and neck cancers. The findings may have implications for the rational use of DNA damaging agents in human cancer.     

Method for Primary Screening of Pharmaceuticals on the Paramecium caudatum Eukaryotic Cell Model

Doklady Biochemistry and Biophysics - The  effect  of  adrenaline  in  various  concentrations  and  dopamine  at...     

Complete genome sequence of Coriobacterium glomerans type strain (PW2T) from the midgut of Pyrrhocoris apterus L. (red soldier bug)

Coriobacterium glomerans Haas and König 1988, is the only species of the genus Coriobacterium, family Coriobacteriaceae, order Coriobacteriales, phylum Actinobacteria. The bacterium thrives as an endosymbiont of pyrrhocorid bugs, i.e. the red fire bug Pyrrhocoris apterus L. The rationale for sequencing the genome of strain PW2^T is its endosymbiotic life style which is rare among members of Actinobacteria. Here we describe the features of this symbiont, together with the complete genome sequence and its annotation. This is the first complete genome sequence of a member of the genus Coriobacterium and the sixth member of the order Coriobacteriales for which complete genome sequences are now available. The 2,115,681 bp long single replicon genome with its 1,804 protein-coding and 54 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.     

Composition of the medial and posterior articular nerves of the mouse knee joint

The number and the distribution of fiber size in the medial (MAN) and posterior (PAN) articular nerves of the mouse knee joint were studied by electron microscopy. The MAN contained 75 &#45 28 nerve fibers consisting of 63 &#45 24 unmyelinated and 12 &#45 6 myelinated fibers. The PAN was composed of 195 &#45 50 nerve fibers, namely 129 &#45 28 unmyelinated and 66 &#45 24 myelinated fibers. A skewed unimodal distribution of the unmyelinated nerve fiber diameters was seen in both nerves ranging from 0.1 to 1.2 &#119 m with a maximum between 0.3 and 0.6 &#119 m. The myelinated nerve fibers in the MAN ranged from 1 to 8 &#119 m with a peak between 2 and 5 &#119 m. In the PAN, their diameters ranged from 1 to 12 &#119 m with a clearly visible peak at 4-5 &#119 m and a plateau at 8-9 &#119 m that may represent a second maximum. These data show that the knee joint innervation of the mouse is comparable to those of the cat and rat concerning the types of nerve fibers and the composition of the two nerves. However, in relation to the much smaller area of tissue to be innervated the total number of primary afferents is considerable smaller in the mouse.     

Complete genome sequence of Nitrosomonas sp. Is79, an ammonia oxidizing bacterium adapted to low ammonium concentrations

Nitrosomonas sp. Is79 is a chemolithoautotrophic ammonia-oxidizing bacterium that belongs to the family Nitrosomonadaceae within the phylum Proteobacteria. Ammonia oxidation is the first step of nitrification, an important process in the global nitrogen cycle ultimately resulting in the production of nitrate. Nitrosomonas sp. Is79 is an ammonia oxidizer of high interest because it is adapted to low ammonium and can be found in freshwater environments around the world. The 3,783,444-bp chromosome with a total of 3,553 protein coding genes and 44 RNA genes was sequenced by the DOE-Joint Genome Institute Program CSP 2006.     

Wide-Ranging Effects of the Yeast Ptc1 Protein Phosphatase Acting Through the MAPK Kinase Mkk1

The Saccharomyces cerevisiae type 2C protein phosphatase Ptc1 is required for a wide variety of cellular functions, although only a few cellular targets have been identified. A genetic screen in search of mutations in protein kinase–encoding genes able to suppress multiple phenotypic traits caused by the ptc1 deletion yielded a single gene, MKK1, coding for a MAPK kinase (MAPKK) known to activate the cell-wall integrity (CWI) Slt2 MAPK. In contrast, mutation of the MKK1 paralog, MKK2, had a less significant effect. Deletion of MKK1 abolished the increased phosphorylation of Slt2 induced by the absence of Ptc1 both under basal and CWI pathway stimulatory conditions. We demonstrate that Ptc1 acts at the level of the MAPKKs of the CWI pathway, but only the Mkk1 kinase activity is essential for ptc1 mutants to display high Slt2 activation. We also show that Ptc1 is able to dephosphorylate Mkk1 in vitro. Our results reveal the preeminent role of Mkk1 in signaling through the CWI pathway and strongly suggest that hyperactivation of Slt2 caused by upregulation of Mkk1 is at the basis of most of the phenotypic defects associated with lack of Ptc1 function.     

The Importance of cGMP Signaling in Sensory Cilia for Body Size Regulation in Caenorhabditis elegans

The body size of Caenorhabditis elegans is thought to be controlled by sensory inputs because many mutants with sensory cilium structure defects exhibit small body size. The EGL-4 cGMP-dependent protein kinase acts in sensory neurons to reduce body size when animals fail to perceive sensory signals. In addition to body size control, EGL-4 regulates various other behavioral and developmental pathways, including those involved in the regulation of egg laying and chemotaxis behavior. Here we have identified gcy-12, which encodes a receptor-type guanylyl cyclase, as a gene involved in the sensory regulation of body size. Analyses with GFP fusion constructs showed that gcy-12 is expressed in several sensory neurons and localizes to sensory cilia. Genetic analyses indicated that GCY-12 acts upstream of EGL-4 in body size control but does not affect other EGL-4 functions. Our studies indicate that the function of the GCY-12 guanylyl cyclase is to provide cGMP to the EGL-4 cGMP-dependent kinase only for limited tasks including body size regulation. We also found that the PDE-2 cyclic nucleotide phosphodiesterase negatively regulates EGL-4 in controlling body size. Thus, the cGMP level is precisely controlled by GCY-12 and PDE-2 to determine body size through EGL-4, and the defects in the sensory cilium structure may disturb the balanced control of the cGMP level. The large number of guanylyl cyclases encoded in the C. elegans genome suggests that EGL-4 exerts pleiotropic effects by partnering with different guanylyl cyclases for different downstream functions.