ARD1 stabilization of TSC2 suppresses tumorigenesis via the mTOR signaling pathway

Mammalian target of rapamycin (mTOR) plays an important role in regulating various cellular functions, and the tuberous sclerosis 1 (TSC1)/TSC2 complex serves as a major repressor of the mTOR pathway. Here we demonstrated that arrest-defective protein 1 (ARD1) physically interacts with, acetylates, and stabilizes TSC2, thereby reducing mTOR activity. The inhibition of mTOR by ARD1 suppresses cell proliferation and increases autophagy, which further impairs tumorigenicity. Correlation between the levels of ARD1 and TSC2 was found in multiple tumor types, suggesting the physiological importance of ARD1 in stabilizing TSC2. Moreover, evaluation of loss of heterozygosity (LOH) at Xq28 revealed allelic loss in 31% of tested breast cancer cell lines and tumor samples. Together, our findings suggest that ARD1 functions as a negative regulator of the mTOR pathway and that dysregulation of the ARD1/TSC2/mTOR axis may contribute to cancer development. To further explore the signaling pathway of ARD1, we provided evidence showing the phosphorylation of ARD1 by IKKβ, which mediated the destabilization of ARD1. Future work may be needed to study the biological effect of this post-translational modification. ^

Identification and evaluation of candidate genes for inherited retinal -degenerative diseases

Although more than 100 genes associated with inherited retinal disease have been mapped to chromosomal locations, less than half of these genes have been cloned. This text includes identification and evaluation of candidate genes for three autosomal dominant forms of inherited retinal degeneration: atypical vitelliform macular dystrophy (VMD1), cone-rod dystrophy (CORD), and retinitis pigmentosa (RP). ^ VMD1 is a disorder characterized by complete penetrance but extremely variable expressivity, and includes macular or peripheral retinal lesions and peripappilary abnormalitites. In 1984, linkage was reported between VMD1 and soluble glutamate-pyruvate transaminase GPT); however, placement of GPT to 8q24 on linkage maps had been debated, and VMD1 did not show linkage to microsatellite markers in that region. This study excluded linkage between the loci by cloning GPT, identifying the nucleotide substitution associated with the GPT sozymes, and by assaying VMD1 family samples with an RFLP designed to detect the substitution. In addition, linkage of VMD1 to the known dominant macular degeneration loci was excluded. ^ CORD is characterized by early onset of color-vision deficiency, and decreased visual acuity, However, this retinal degeneration progresses to no light perception, severe macular lesion, and “bone-spicule” accumulations in the peripheral retina. In this study, the disorder in a large Texan family was mapped to the CORD2 locus of 19q13, and a mutation in the retina/pineal-specific cone-rod homeobox gene (CRX) was identified as the disease cause. In addition, mutations in CRX were associated with significantly different retinal disease phenotypes, including retinitis pigmentosa and Leber congenital amaurosis. ^ Many of the mutations leading to inherited retinal disorders have been identified in genes like CRX, which are expressed predominantly in the retina and pineal gland. Therefore, a combination of database analysis and laboratory investigation was used to identify 26 novel retina/pineal-specific expressed sequence tag (EST) clusters as candidate genes for inherited retinal disorders. Eight of these genes were mapped into the candidate regions of inherited retinal degeneration loci. ^ Two of the eight clusters mapped into the retinitis pigmentosa RP13 candidate region of 17p13, and were both determined to represent a single gene that is highly expressed in photoreceptors. This gene, the Ah receptor-interacting like protein-1 (AIPL1), was cloned, characterized, and screened for mutations in RP13 patient DNA samples. ^

Signaling pathways in the transcriptional and post-translational regulation of the human glutathione S -transferase P1 gene

The human glutathione S-transferase P1 (GSTP1) protein is an endogenous inhibitor of c-jun N-terminal kinases (JNKs) and an important phase II detoxification enzyme. ^ Recent identification of a cAMP response element (CRE) in the 5 ′-region of the human GSTP1 gene and several putative phosphorylation sites for the Ser/Thr protein kinases, including, cAMP-dependent protein kinases (PKAs), protein kinases C (PKCs), and JNKs in the GSTP1 protein raised the possibility that signaling pathways may play an important role in the transcriptional and post-translational regulation of GSTP1 gene. This study examined (a) whether the signaling pathway mediated by CAMP, via the GSTP1 CRE, is involved in the transcriptional regulation of the GSTP1 gene, (b) whether signaling pathways mediated by the Ser/Thr protein kinases (PKAs, PKCs, and JNKs) induce post-translational modification, viz. phosphorylation of the GSTP1 protein, and (c) whether such phosphorylation of the GSTP1 protein alters its functions in metabolism and in JNK signaling. ^ The first major finding in this study is the establishment of the human GSTP1 gene as a novel CAMP responsive gene in which transcription is activated via an interaction between PKA activated CRE binding protein-1 (CREB-1) and the CRE in the 5′-regulatory region. ^ The second major finding in this study is the observation that the GSTP1 protein undergoes phosphorylation and functionally activated by second messenger-activated protein kinases, PKA and PKC, in tumor cells with activated signaling pathways. Following phosphorylation by PKA or PKC, the catalytic activity of the GSTP1 protein was significantly enhanced, as indicated by a decrease in its Km (2- to 3.6-fold) and an increase in Kcat/ Km (1.6- to 2.5-fold) for glutathione. Given the frequent over-expression of GSTP1 and the aberrant PKA/PKC signaling cascade observed in tumors, these findings suggest that phosphorylation of GSTP1 may contribute to the malignant progression and drug-resistant phenotype of these tumors. ^ The third major finding in this study is that the GSTP1 protein, an inhibitor of JNKs, undergoes significant phosphorylation in tumor cells with activated JNK signaling pathway and in those under oxidative stress. Following phosphorylation by JNK, the ability of GSTP1 to inhibit JNK downstream function, i.e. c-jun phosphorylation, was significantly enhanced, suggesting a feedback mechanism of regulation of JNK-mediated cellular signaling. (Abstract shortened by UMI.) ^

(Table 2) Concentration of phytoplankton pigments in Adventfjorden in spring and summer of 1997

The study was carried out from April 30 until July 13 of 1997 in Adventfjorden (Spitsbergen). Formation of a less saline and warmer surface water (~1 m thick) caused by melting of the ice was observed in the fjord during the first days of May. In summer the less saline surface layer was about 3 m thick. Euphotic depth measured under the ice sheet reached 12 m, whereas load of mineral matter brought with riverine discharge in summer (content of total particulate matter in the fjord reached 1.66 kg/m**2) dramatically reduced euphotic zone depth to 0.35 m. By pigment measurement three phases of phytoplankton development in Adventfjorden were distinguished: (1) spring bloom that has started under fast ice and reached maximum in the mid of May, (2) stagnation period in June, (3) increase of pigment concentration in July, what could indicate start of the next algae bloom. Analyses of chlorophylls and carotenoids revealed that diatoms (chl c, fucoxanthin), and green algae (chl b, lutein) dominated phytoplankton community in the fjord. Moreover, presence of peridinin indicates presence of Dinophyta and alloxanthin - occurence of Cryptophyta. In May and June 1997 phytoplankton appeared mainly in the surface of water, while in July, as a result of inflow of turbulent riverine waters into Adventfjorden, algae cells were pushed down and the highest numbers were observed at depth ~20 m. Great phaeopigments to chl a ratio (= 0.54) found in fjord seston in June and July probably shows strong impact of zooplankton grazing on phytoplankton development. High contribution of chlorophyllide a in porphyrin a poll in samples collected under fast ice (chlorophyllide a / chl a ratio = 0.18) reflects the final stage of algal communitie succession in ice, just before spring ice melt and release of biota to oceanic water. Chlorophyllide a content during summer was minor or not detectable, demonstrating that diatom cells were in good physiological condition. High chl a allomer / chl a ratio (average = 0.11 for the period investigated) confirms high oxygen concentration in environment of Adventfjorden.

Phototrophic carbon fixation rates measured in water samples from the Porcupine Abyssal Plains during James Cook cruise JC087

Water samples were collected from pre-dawn CTD casts at 5 depths corresponding to 55%, 20%, 7%, 5% and 1% of surface irradiance. 1 litre water samples wrapped with optical filters to replicate light levels. Spiked with 200 µL of 13C labelled sodium bicarbonate. After 24 hourse filtered through ashed 25mm GF/F (Whatman) filters, rinsed with HCl solution (1-2%) and stored at -20oC. On shore encapsulated in tin capsules and analysed for 13C isotopic enrichment. Carbon uptake rates calculated using the equations of Fernandez et al. (2005).

Global Lithological Map Database v1.0 (gridded to 0.5° spatial resolution)

Lithology describes the geochemical, mineralogical, and physical properties of rocks. It plays a key role in many processes at the Earth surface, especially the fluxes of matter to soils, ecosystems, rivers, and oceans. Understanding these processes at the global scale requires a high resolution description of lithology. A new high resolution global lithological map (GLiM) was assembled from existing regional geological maps translated into lithological information with the help of regional literature. The GLiM represents the rock types of the Earth surface using 1,235,400 polygons. The lithological classification consists of three levels. The first level contains 16 lithological classes comparable to previously applied definitions in global lithological maps. The additional two levels contain 12 and 14 subclasses, respectively, which describe more specific rock attributes. According to the GLiM, the Earth is covered by 64 % sediments (a third of which is carbonates), 13 % metamorphics, 7 % plutonics, and 6 % volcanics, and 10% are covered by water or ice. The high resolution of the GLiM allows observation of regional lithological distributions which often vary from the global average. The GLiM enables regional analysis of Earth surface processes at global scales.

Table 3: Comparison of freshwater species in Ob and Yenisei during the 1997 cruise

Phytoplankton biomass distribution (chlorophyll a, chl. a) and species composition (cell numbers) were investigated during three expeditions to the Kara Sea with "Akademik Boris Petrov" (BP) in 1997, 1999, and 2000. The distribution of biomass in the estuaries of Ob and Yenisei showed a similar range in 1997 (0.2 to 3.2 µg/l) and 2000 (0.4 to 3.5 ug/l); higher chl. a concentrations during these two years were found in Yenisei than in Ob. In 1999, phytoplankton biomass in the Ob and Ob Estuary was much higher than in 1997 and 2000, with maximum values above 10.0 ug chl. a/l. In 1999, biomass in Yenisei was lower (1.5 to ~5 ug/l) than in Ob but slightly higher than in 1997 and in 2000. During the expedition in 2000, the research area extended farther to the north, here, lowest phytoplankton biomass during all three years was found. Typical summer values for integrated chl.a biomass (surface to bottom) ranged between 6 and 20 mg m**-2. Strong differences existed in species composition in both rivers, the estuaries, and the open Kara Sea. In general, three or four different populations could be distinguished in surface waters: (1) freshwater diatoms together with bluegreen algae in both rivers, (2) centric and small pennate diatoms mainly brackish species in the estuaries, (3) north of 74°N, brackish/marine species dominated, i.e. in 1999 Thalassiosira cfpunctigera and Chaetoceros spp prevailed in the phytoplankton bloom in Ob. (4) At the northernmost, almost marine stations, a region with a more heterogeneous composition of unicellular plankton was encountered. We assume, we found different seasonal signals of phytoplankton development during 1997/2000 and 1999, respectively. However, the yearly fluctuation of freshwater runoff of both rivers seems to have the strongest influence on the timing and duration of phytoplankton blooms, species compositions and biomass standing stocks during summer.

Vertical distribution of large particulate matter in the Western Mediterranean Sea

The relationship between mesoscale hydrodynamics and the distribution of large particulate matter (LPM, particles larger than 200 ?m) in the first 1000 m of the Western Mediterranean basin was studied with a microprocessor-driven CTD-video package, the Underwater Video Profiler (UVP). Observations made during the last decade showed that, in late spring and summer, LPM concentration was high in the coastal part of the Western Mediterranean basin at the shelf break and near the continental slope (computed maximum: 149 ?g C/l between 0 and 100 m near the Spanish coast of the Gibraltar Strait). LPM concentration decreased further offshore into the central Mediterranean Sea where, below 100 m, it remained uniformly low, ranging from 2 to 4 ?g C/l. However, a strong variability was observed in the different mesoscale structures such as the Almeria-Oran jet in the Alboran Sea or the Algerian eddies. LPM concentration was up to one order of magnitude higher in fronts and eddies than in the adjacent oligotrophic Mediterranean waters (i.e. 35 vs. 8 ?g C/l in the Alboran Sea or 16 vs. 3 ?g C/l in a small shear cyclonic eddy). Our observations suggest that LPM spatial heterogeneity generated by the upper layer mesoscale hydrodynamics extends into deeper layers. Consequently, the superficial mesoscale dynamics may significantly contribute to the biogeochemical cycling between the upper and meso-pelagic layers.

Interaction of SP100 with HP1 proteins: A link between the promyelocytic leukemia-associated nuclear bodies and the chromatin compartment

The PML/SP100 nuclear bodies (NBs) were first described as discrete subnuclear structures containing the SP100 protein. Subsequently, they were shown to contain the PML protein which is part of the oncogenic PML-RARα hybrid produced by the t(15;17) chromosomal translocation characteristic of acute promyelocytic leukemia. Yet, the physiological role of these nuclear bodies remains unknown. Here, we show that SP100 binds to members of the heterochromatin protein 1 (HP1) families of non-histone chromosomal proteins. Further, we demonstrate that a naturally occurring splice variant of SP100, here called SP100-HMG, is a member of the high mobility group-1 (HMG-1) protein family and may thus possess DNA-binding potential. Both HP1 and SP100-HMG concentrate in the PML/SP100 NBs, and overexpression of SP100 leads to enhanced accumulation of endogenous HP1 in these structures. When bound to a promoter, SP100, SP100-HMG and HP1 behave as transcriptional repressors in transfected mammalian cells. These observations present molecular evidence for an association between the PML/SP100 NBs and the chromatin nuclear compartment. They support a model in which the NBs may play a role in certain aspects of chromatin dynamics.

Antiviral agent blocks breathing of the common cold virus

A dynamic capsid is critical to the events that shape the viral life cycle; events such as cell attachment, cell entry, and nucleic acid release demand a highly mobile viral surface. Protein mass mapping of the common cold virus, human rhinovirus 14 (HRV14), revealed both viral structural dynamics and the inhibition of such dynamics with an antiviral agent, WIN 52084. Viral capsid digestion fragments resulting from proteolytic time-course experiments provided structural information in good agreement with the HRV14 three-dimensional crystal structure. As expected, initial digestion fragments included peptides from the capsid protein VP1. This observation was expected because VP1 is the most external viral protein. Initial digestion fragments also included peptides belonging to VP4, the most internal capsid protein. The mass spectral results together with x-ray crystallography data provide information consistent with a “breathing” model of the viral capsid. Whereas the crystal structure of HRV14 shows VP4 to be the most internal capsid protein, mass spectral results show VP4 fragments to be among the first digestion fragments observed. Taken together this information demonstrates that VP4 is transiently exposed to the viral surface via viral breathing. Comparative digests of HRV14 in the presence and absence of WIN 52084 revealed a dramatic inhibition of digestion. These results indicate that the binding of the antiviral agent not only causes local conformational changes in the drug binding pocket but actually stabilizes the entire viral capsid against enzymatic degradation. Viral capsid mass mapping provides a fast and sensitive method for probing viral structural dynamics as well as providing a means for investigating antiviral drug efficacy.

A human homolog of the Saccharomyces cerevisiae REV3 gene, which encodes the catalytic subunit of DNA polymerase ζ

To get a better understanding of mutagenic mechanisms in humans, we have cloned and sequenced the human homolog of the Saccharomyces cerevisiae REV3 gene. The yeast gene encodes the catalytic subunit of DNA polymerase ζ, a nonessential enzyme that is thought to carry out translesion replication and is responsible for virtually all DNA damage-induced mutagenesis and the majority of spontaneous mutagenesis. The human gene encodes an expected protein of 3,130 residues, about twice the size of the yeast protein (1,504 aa). The two proteins are 29% identical in an amino-terminal region of ≈340 residues, 39% identical in a carboxyl-terminal region of ≈850 residues, and 29% identical in a 55-residue region in the middle of the two genes. The sequence of the expected protein strongly predicts that it is the catalytic subunit of a DNA polymerase of the pol ζ type; the carboxyl-terminal domain possesses, in the right order, the six motifs characteristic of eukaryotic DNA polymerases, most closely resembles yeast pol ζ among all polymerases in the GenBank database, and is different from the human α, δ, and ɛ enzymes. Human cells expressing high levels of an hsREV3 antisense RNA fragment grow normally, but show little or no UV-induced mutagenesis and are slightly more sensitive to killing by UV. The human gene therefore appears to carry out a function similar to that of its yeast counterpart.

ActA is a dimer

ActA, a surface protein of Listeria monocytogenes, is able to induce continuous actin polymerization at the rear of the bacterium, in the cytosol of the infected cells. Its N-terminal domain is sufficient to induce actin tail formation and movement. Here, we demonstrate, using the yeast two-hybrid system, that the N-terminal domain of ActA may form homodimers. By using chemical cross-linking to explore the possibility that ActA could be a multimer on the surface of the bacteria, we show that ActA is a dimer. Cross-linking experiments on various L. monocytogenes strains expressing different ActA variants demonstrated that the region spanning amino acids 97–126, and previously identified as critical for actin tail formation, is also critical for dimer formation. A model of actin polymerization by L. monocytogenes, involving the ActA dimer, is presented.