Morphology and dynamics of the jets of comet 67P/Churyumov-Gerasimenko: Early-phase development

Aims. The OSIRIS camera onboard the Rosetta spacecraft obtained close-up views of the dust coma of comet 67P. The jet structures can be used to trace their source regions and to examine the possible effect of gas-surface interaction. Methods. We analyzed the wide-angle images obtained in the special dust observation sequences between August and September 2014. The jet features detected in different images were compared to study their time variability. The locations of the potential source regions of some of the jets are identified by ray tracing. We used a ring-masking technique to calculate the brightness distribution of dust jets along the projected distance. Results. The jets detected between August and September 2014 mostly originated in the Hapi region. Morphological changes appeared over a timescale of several days in September. The brightness slope of the dust jets is much steeper than the background coma. This might be related to the sublimation or fragmentation of the emitted dust grains. Interaction of the expanding gas flow with the cliff walls on both sides of Hapi could lead to erosion and material down-fall to the nucleus surface.

Scientific assessment of the quality of OSIRIS images

Context. OSIRIS, the scientific imaging system onboard the ESA Rosetta spacecraft, has been imaging the nucleus of comet 67P/Churyumov-Gerasimenko and its dust and gas environment since March 2014. The images serve different scientific goals, from morphology and composition studies of the nucleus surface, to the motion and trajectories of dust grains, the general structure of the dust coma, the morphology and intensity of jets, gas distribution, mass loss, and dust and gas production rates. Aims. We present the calibration of the raw images taken by OSIRIS and address the accuracy that we can expect in our scientific results based on the accuracy of the calibration steps that we have performed. Methods. We describe the pipeline that has been developed to automatically calibrate the OSIRIS images. Through a series of steps, radiometrically calibrated and distortion corrected images are produced and can be used for scientific studies. Calibration campaigns were run on the ground before launch and throughout the years in flight to determine the parameters that are used to calibrate the images and to verify their evolution with time. We describe how these parameters were determined and we address their accuracy. Results. We provide a guideline to the level of trust that can be put into the various studies performed with OSIRIS images, based on the accuracy of the image calibration.

Growth Cone Localization of the mRNA Encoding the Chromatin Regulator HMGN5 Modulates Neurite Outgrowth

Neurons exploit local mRNA translation and retrograde transport of transcription factors to regulate gene expression in response to signaling events at distal neuronal ends. Whether epigenetic factors could also be involved in such regulation is not known. We report that the mRNA encoding the high-mobility group N5 (HMGN5) chromatin binding protein localizes to growth cones of both neuron-like cells and of hippocampal neurons, where it has the potential to be translated, and that HMGN5 can be retrogradely transported into the nucleus along neurites. Loss of HMGN5 function induces transcriptional changes and impairs neurite outgrowth, while HMGN5 overexpression induces neurite outgrowth and chromatin decompaction; these effects are dependent on growth cone localization of Hmgn5 mRNA. We suggest that the localization and local translation of transcripts coding for epigenetic factors couple the dynamic neuronal outgrowth process with chromatin regulation in the nucleus.

ALTERATIONS IN HYPOTHALAMIC CONTENT OF LUTEINIZING HORMONE RELEASING HORMONE AND PITUITARY RESPONSIVENESS ASSOCIATED WITH TESTICULAR REGRESSION INDUCED BY PINEAL RELATED TREATMENTS IN THE GOLDEN HAMSTER

The adult male golden hamster, when exposed to blinding (BL), short photoperiod (SP), or daily melatonin injections (MEL) demonstrates dramatic reproductive collapse. This collapse can be blocked by removal of the pineal gland prior to treatment. Reproductive collapse is characterized by a dramatic decrease in both testicular weight and serum gonadotropin titers. The present study was designed to examine the interactions of the hypothalamus and pituitary gland during testicular regression, and to specifically compare and contrast changes caused by the three commonly employed methods of inducing testicular regression (BL,SP,MEL). Hypothalamic LHRH content was altered by all three treatments. There was an initial increase in content of LHRH that occurred concomitantly with the decreased serum gonadotropin titers, followed by a precipitous decline in LHRH content which reflected the rapid increases in both serum LH and FSH which occur during spontaneous testicular recrudescence. In vitro pituitary responsiveness was altered by all three treatments: there was a decline in basal and maximally stimulatable release of both LH and FSH which paralleled the fall of serum gonadotropins. During recrudescence both basal and maximal release dramatically increased in a manner comparable to serum hormone levels. While all three treatments were equally effective in their ability to induce changes at all levels of the endocrine system, there were important temporal differences in the effects of the various treatments. Melatonin injections induced the most rapid changes in endocrine parameters, followed by exposure to short photoperiod. Blinding required the most time to induce the same changes. This study has demonstrated that pineal-mediated testicular regression is a process which involves dynamic changes in multiply-dependent endocrine relationships, and proper evaluation of these changes must be performed with specific temporal events in mind. ^

CHOLINERGIC MODULATION OF THALAMIC INPUT INTO THE CINGULATE CORTEX

This research demonstrates cholinergic modulation of thalamic input into the limbic cortex. A projection from the mediodorsal thalamus (MD) to the anterior cingulate cortex was defined anatomically and physiologically. Injections of horse-radish peroxidase into the anterior cingulate cortex labels neurons in the lateral, parvocellular, region of MD. Electrical Stimulation of this area produces a complex field potential in the anterior cingulate cortex which was further characterized by current density analysis and single cell recordings.^ The monsynaptic component of the response was identified as a large negative field which is maximal in layer IV of the anterior cingulate cortex. This response shows remarkable tetanic potentiation of frequencies near 7 Hz. During a train of 50 or more stimuli, the response would grow quickly and remain at a fairly stable potentiated level throughout the train.^ Cholinergic modulation of this thalamic response was demonstrated by iontophoretic application of the cholinergic agonist carbachol decreased the effectiveness of the thalamic imput by rapidly attenuation the response during a train of stimuli. The effect was apparently mediated by muscarinic receptors since the effect of carbachol was blocked by atropine but not by hexamethonium.^ To determine the source of the cingulate cortex cholinergic innervation, lesions were made in the anterior and medial thalamus and in the nucleus of the diagonal band of Broca. The effects of these lesions on choline acetyltranferase activity in the cingulate cortex were determined by a micro-radio-enzymatical assay. Only the lesions of the nucleus of the diagonal band significantly decreased the choline acetyltransferase activity in the cingulate cortex regions. Therefore, the diagonal band appears to be a major source of sensory cholinergic innervation and may be involved in gating of sensory information from the thalamus into the limbic cortex. Attempts to modulate the cingulate response to MD stimulation with electrical stimulation of the diagonal band, however were not successful.^

Mapping and characterization of the Xlsirt P11 RNA cis-acting localization element

In many organisms, polarity of the oocyte is established post-transcriptionally via subcellular RNA localization. Many RNAs are localized during oogenesis in Xenopus laevis, including Xlsirts ( Xenopus laevis short interspersed repeat transcripts) [Kloc, 1993]. Xlsirts constitute a large family defined by highly homologous repeat units 79–81 nucleotides in length. Endogenous Xlsirt RNAs use the METRO (Message Transport Organizer) pathway of localization, where RNAs are transported from the nucleus to the mitochondrial cloud in stage I oocytes. Secondly, RNAs anchor at the vegetal pole in stage II oocytes. Exogenous Xlsirt RNAs can also utilize the Late pathway of localization, which involves localization to the vegetal cortex during stage III of oogenesis and results in RNAs anchored in the cortex of the entire vegetal hemisphere. ^ The Xlsirts localization signal is contained within the repeat region. This study was designed to test the hypothesis that there are cis -acting localization elements in Xlsirts, and that higher order structure plays a role. Results of experiments on Xlsirt P11, a 1700 basepair (bp) family member, led to the conclusion that a 137-bp fragment of the repetitive region is necessary and sufficient for METRO and Late pathway localization. This analysis definitively demonstrates that the Xlsirt localization signal for the METRO and Late pathways reside within the repetitive region and not within the flanking regions. Analysis of Xlsirt linker scanning mutations revealed two METRO-pathway specific subelements, and one Late-pathway specific subelement. Functional, computer, and biochemical evidence relates the higher order structure of this element to its ability to function as a localization element. ^ Xlsirt 137 is 99% identical to the Xlsirt consensus sequence identified in this study, suggesting that it is the localization element for all localized Xlsirt family members. The repeat unit was reframed based on function, rather than arbitrarily based on sequence. This work supports the hypothesis presented in 1981 by George Spohr, who originally isolated the Xlsirts, which stated that the highly conserved repetitive elements must be constrained from variability due to some unknown function of the repeats themselves. These studies shed light on the mechanism of RNA localization, linking structure and function. ^

RNA binding characteristics and overall topology of the vaccinia virus polyadenylation complex

mRNA 3′ polyadenylation is central to mRNA biogenesis in prokaryotes and eukaryotes, and is implicated in numerous aspects of mRNA metabolism, including efficiency of mRNA export from the nucleus, message stability, and initiation of translation. However, due to the great complexity of the eukaryotic polyadenylation apparatus, the mechanisms of RNA 3 ′ end processing have remained elusive. Although the RNA processing reactions leading to polyadenylated messenger RNA have been studied in many systems, and much progress has been made, a complete understanding of the biochemistry of the poly(A) polymerase enzyme is still lacking. My research uses Vaccinia virus as a model system to gain a better understanding of this complicated polyadenylation process, which consist of RNA binding, catalysis and polymerase translocation. ^ Vaccinia virus replicates in the cytoplasm of its host cell, so it must employ its own poly(A) polymerase (PAP), a heterodimer of two virus encoded proteins, VP55 and VP39. VP55 is the catalytic subunit, adding 30 adenylates to a non-polyadenylated RNA in a rapid processive manner before abruptly changing to a slow, non-processive mode of adenylate addition and dissociating from the RNA. VP39 is the stimulatory subunit. It has no polyadenylation catalytic activity by itself, but when associated with VP55 it facilitates the semi-processive synthesis of tails several hundred adenylates in length. ^ Oligonucleotide selection and competition studies have shown that the heterodimer binds a minimal motif of (rU)2 (N)25 U, the “heterodimer binding motif”, within an oligonucleotide, and its primer selection for polyadenylation is base-type specific. ^ Crosslinking studies using photosensitive uridylate analogs show that within a VP55-VP39-primer ternary complex, VP55 comes into contact with all three required uridylates, while VP39 only contacts the downstream uridylate. Further studies, using a backbone-anchored photosensitive crosslinker show that both PAP subunits are in close proximity to the downstream −10 to −21 region of 50mer model primers containing the heterodimer binding motif. This equal crosslinking to both subunits suggests that the dimerization of VP55 and VP39 creates either a cleft or a channel between the two subunits through which this region of RNA passes. ^ Peptide mapping studies of VP39 covalently crosslinked to the oligonucleotide have identified residue R107 as the amino acid in close proximity to the −10 uridylate. This helps us project a conceptual model onto the known physical surface of this subunit. In the absence of any tertiary structural data for VP55, we have used a series of oligonucleotide selection assays, as well as crosslinking, nucleotide transfer assays, and gel shift assays to gain insight into the requirements for binding, polyadenylation and translocation. Collectively, these data allow us to put together a comprehensive model of the structure and function of the polyadenylation ternary complex consisting of VP39, VP55 and RNA. ^

Structural determinants of subunit -subunit interactions and subcellular localization of the calcium /calmodulin -dependent protein kinase II

The multifunctional Ca$\sp{2+}$/calmodulin-dependent protein kinase II (CaM kinase) is a Ser/Thr directed protein kinase that participates in diverse Ca$\sp{2+}$ signaling pathways in neurons. The function of CaM kinase depends upon the ability of subunits to form oligomers and to interact with other proteins. Oligomerization is required for autophosphorylation which produces significant functional changes that include Ca$\sp{2+}$/calmodulin-independent activity and calmodulin trapping. Associations with other proteins localize CaM kinase to specific substrates and effectors which serves to optimize the efficiency and speed of signal transduction. In this thesis, we investigate the interactions that underlie the appropriate positioning of CaM kinase activity in cells. We demonstrate that the subcellular distribution of CaM kinase is dynamic in hippocampal slices exposed to anoxic/aglycemic insults and to high K$\sp{+}$-induced depolarization. We determine the localization of CaM kinase domains expressed in neurons and PC-12 cells and find that the C-terminal domain of the $\alpha$ subunit is necessary for localization to dendrites. Moreover, monomeric forms of the enzyme gain access to the nucleus. Attempts made to identify novel CaM kinase binding proteins using the yeast two-hybrid system resulted in the isolation of hundreds of positive clones. Those that have been sequenced are identical to CaM kinase isoforms. Finally, we report the discovery of specific regions within the C-terminal domain that are necessary and sufficient for subunit-subunit interactions. Differences between the $\alpha$ and $\beta$ isoforms were discovered that indicate unique structural requirements for oligomerization. A model for how CaM kinase subunits interact to form holoenzymes and how structural heterogeneity might influence CaM kinase function is presented. ^

The role of Ski/Sno oncoproteins as negative regulators of the TGF-beta/SMAD signaling pathway in B -cell non -Hodgkin's lymphoma (NHL-B)

Non-Hodgkin's lymphomas are common tumors of the human immune system, primarily of B cell lineage (NHL-B). Negative growth regulation in the B cell lineage is mediated primarily through the TGF-β/SMAD signaling pathway that regulates a variety of tumor suppressor genes. Ski was originally identified as a transforming oncoprotein, whereas SnoN is an isoform of the Sno protein that shares a large region of homology with Ski. In this study, we show that Ski/SnoN are endogenously over-expressed both in patients' lymphoma cells and NHL-B cell lines. Exogenous TGF-β1 treatment induces down-regulation of Ski and SnoN oncoprotein expression in an NHL-B cell line, implying that Ski and SnoN modulate the TGF-β signaling pathway and are involved in cell growth regulation. Furthermore, we have developed an NHL-B cell line (DB) that has a null mutation in TGF-β receptor type II. In this mutant cell line, Ski/SnoN proteins are not down-regulated in response to TGF-β1 treatment, suggesting that downregulation of Ski and SnoN proteins in NHL-B require an intact functional TGF-β signaling pathway Resting normal B cells do not express Ski until activated by antigens and exogenous cytokines, whereas a low level of SnoN is also present in peripheral blood Go B cells. In contrast, autonomously growing NHL-B cells over-express Ski and SnoN, implying that Ski and SnoN are important cell cycle regulators. To further investigate a possible link between reduction of the Ski protein level and growth inhibition, Ski antisense oligodeoxynucleotides were transfected into NHL-B cells. The Ski protein level was found to decrease to less than 40%, resulting in restoring the effect of TGF-β and leading to cell growth inhibition and G1 cell cycle arrest. Co-immunoprecipitation experiments demonstrated that Ski associates with Smad4 in the nucleus, strongly suggesting that over-expression of the nuclear protein Ski and/or SnoN negatively regulates the TGF-β pathway, possibly by modulating Smad-mediated tumor suppressor gene expression. Together, in NHL-B, the TGF-β/SMAD growth inhibitory pathway is usually intact, but over-expression of the Ski and/or SnoN, which binds to Smad4, abrogates the negative regulatory effects of TGF-β/SMAD in lymphoma cell growth and potentiates the growth potential of neoplastic B cells. ^

The role of the conserved N -terminal domain of STAT3 in STAT3 dephosphorylation and nuclear export

Rapid redistribution of STAT subcellular localization is an essential feature of cytokine signaling. To elucidate the molecular basis of STAT3 function, which plays a critical role in controlling innate immune responses in vivo, we initiated studies to determine the mechanisms controlling STAT3 nuclear trafficking. We found that STAT3 is transported to the nucleus in the absence of cytokine treatment, as judged by indirect immunofluorescence studies in the presence of leptomycin B, an inhibitor of CRM1-dependent nuclear export, suggesting that the non-phosphorylated STAT3 protein contains a functional nuclear import signal. An isoform lacking the STAT3 N-terminal domain (Δ133STAT3) retains the ability to undergo constitutive nuclear localization, indicating that this region is not essential for cytokine-independent nuclear import. Δ133STAT3 is also transported to the nucleus following stimulation with interleukin-6 (IL-6). Interestingly, IL-6-dependent tyrosine phosphorylation of Δ133STAT3 appears to be prolonged and the nuclear export of the protein delayed in cells expressing endogenous STAT3, consistent with defective Δ133STAT3 dephosphorylation. Endogenous STAT3 does not promote the nuclear export of Δ133STAT3, although dimerization between endogenous Stat3 and Δ133STAT3 is detected readily. Thus, the STAT3 N-terminal domain is not required for dimerization with full-length STAT3, yet appears to play a role in proper export of Stat3 from the nucleus following cytokine stimulation. STAT3-deficient cells reconstituted with Δ133STAT3 show enhanced and prolonged Stat1 signaling in response to IL-6, suggesting that induction of the STAT3-dependent negative regulator SOCS3 is impaired. In fact, Δ133STAT3 fails to induce SOCS3 mRNA efficiently. These studies collectively indicate that the STAT3 N-terminal region may be important for IL-6-dependent target gene activation and nuclear dephosphorylation, while dispensable for nuclear import. STAT3 is an oncogene. STAT3 is constitutively activated in primary tumors of many types. Thus far, research in the design of STAT3 protein inhibitors has focused on the SH2 and DNA-binding domains of STAT3. Interference with these domains eliminates all signaling through STAT3. If the N-terminal domain is involved in tetramerization on a subset of target genes, inhibition of this region may lead to a more selective inhibition of some STAT3 functions while leaving others intact. ^

Molluscan biostratigraphy of the Miocene and Chatt in the southern Herzogtum Lauenburg (Schleswig-Holstein)

A study was made of the marine molluscan fauna from 12 borings in the Schwarzenbek area. In the fossil rich facies underlying the 'Braunkohlensande', the Neochatt and Vierland faunal sequences could be described and used to define the Oligocene/Miocene boundary. The Neochatt, defined by Pectinidae, seems to be more closely related to the Miocene than previously thought. Nevertheless, a sufficient number of additional molluscan species are present for placing the Neochatt/Vierland boundary. Overlying the Braunkohlensande, the sandy Reinbek fauna as well as Glimmerton faunas of the Reinbek and Langenfelde stages could be described.

Characterization of the Abydos region through OSIRIS high-resolution images in support of CIVA measurements

Context. On 12 November 2014, the European mission Rosetta delivered the Philae lander on the nucleus of comet 67P /Churyumov-Gerasimenko (67P). After the first touchdown, the lander bounced three times before finally landing at a site named Abydos. Aims. We provide a morphologically detailed analysis of the Abydos landing site to support Philae's measurements and to give context for the interpretation of the images coming from the Comet Infrared and Visible Analyser (CIVA) camera system onboard the lander. Methods. We used images acquired by the OSIRIS Narrow Angle Camera (NAC) on 6 December 2014 to perform the analysis of the Abydos landing site, which provided the geomorphological map, the gravitational slope map, the size-frequency distribution of the boulders. We also computed the albedo and spectral reddening maps. Results. The morphological analysis of the region could suggest that Philae is located on a primordial terrain. The Abydos site is surrounded by two layered and fractured outcrops and presents a 0.02 km(2) talus deposit rich in boulders. The boulder size frequency distribution gives a cumulative power-law index of 4.0 + 0.3/0.4, which is correlated with gravitational events triggered by sublimation and /or thermal fracturing causing regressive erosion. The average value of the albedo is 5.8% at lambda(1) = 480.7 nm and 7.4% at lambda(2) = 649.2 nm, which is similar to the global albedos derived by OSIRIS and CIVA, respectively.