Functional studies of *Ras and Bcl-2 oncoproteins in keratinocyte homeostasis and multistep skin carcinogenesis

To assess the effect of deregulated Ha-ras and bcl-2, individually and in combination on epidermal keratinocyte homeostasis and during multistep skin carcinogenesis, we generated skin-specific transgenic mice and keratinocyte transfectants constitutively expressing oncogenic Ha-ras and bcl-2 proteins. The deregulated Ha-ras and bcl-2 expression contributing to homeostatic imbalances in the skin had an additive effect on the probability of tumor development. They were also cooperative in incidence, growth, and latency of tumor formation, and they exhibited synergistic cooperation in malignant transformation of benign papillomas. To explain the homeostatic imbalances by Ha-ras and bcl-2 overexpression in the skin, we investigated the three major cellular processes of proliferation, cell death, and differentiation. Epidermal expression of Bcl-2 retarded keratinocyte proliferation in the epidermis of neonatal mice compared with results for control littermates. Constitutive expression of Ha-ras increased keratinocyte proliferation, and co-expression of bcl-2 modestly suppressed the ras-mediated abnormal proliferation of neonatal keratinocytes. Bcl-2 proteins in keratinocytes protected UV-treated cells from apoptotic cell death regardless of oncogenic ras expression in both non-neoplastic neonatal epidermis and human keratinocyte cell lines. The spontaneous apoptotic index (AI) was also lower in papillomas constitutively expressing bcl-2 compared with the ones that developed in control mice. Ras-overexpressing epidermis, including that in ras/bcl-2 double transgenic mice, had abnormal differentiation patterns compared with controls. The oncogenic ras protein had alterations in both epidermal distribution and the extent of cytokeratin 14 and involucrin expression. Abnormal expression of the hyperproliferation marker cytokeratin 6 and modest down regulation of cytokeratin 1 were also detected. Late appearance of filaggrin was another abnormal phenotype of the ras-expressing epidermis. Overexpression of bcl-2 had no effect on epidermal differentiation. Together, these findings suggest that constitutive expression of oncogenic Ha-ras and bcl-2 are important determinants of epidermal proliferation, viability and differentiation. In summary, our results demonstrated that the disruption of epidermal homeostasis by overexpressed ras and bcl-2 predisposes to hyperplastic growth of the epidermis and to papilloma development and that these proteins with distinct mechanisms for oncogenesis are functionally synergistic for malignant transformation of chemically induced skin carcinogenesis. ^

Net primary productivity, POC export, Th234 and U238 activities and abundance of autotrophs during U.S.C.G.C. Healy cruises HLY0802 and HLY0803

Seasonal patterns in the partitioning of phytoplankton carbon during receding sea ice conditions in the eastern Bering Sea water column are presented using rates of 14C net primary productivity (NPP), phototrophic plankton carbon content, and POC export fluxes from shelf and slope waters in the spring (March 30-May 6) and summer (July 3-30) of 2008. At ice-covered and marginal ice zone (MIZ) stations on the inner and middle shelf in spring, NPP averaged 76 ± 93 mmol C/m**2/d, and in ice-free waters on the outer shelf NPP averaged 102 ± 137 mmol C/m**2/d. In summer, rates of NPP were more uniform across the entire shelf and averaged 43 ± 23 mmol C/m**2/d over the entire shelf. A concomitant shift was observed in the phototrophic pico-, nano-, and microplankton community in the chlorophyll maximum, from a diatom dominated system (80 ± 12% autotrophic C) in ice covered and MIZ waters in spring, to a microflagellate dominated system (71 ± 31% autotrophic C) in summer. Sediment trap POC fluxes near the 1% PAR depth in ice-free slope waters increased by 70% from spring to summer, from 10 ± 7 mmol C/m**2/d to 17 ± 5 mmol C/m**2/d, respectively. Over the shelf, under-ice trap fluxes at 20 m were higher, averaging 43 ± 17 mmol C/m**2/d POC export over the shelf and slope estimated from 234Th deficits averaged 11 ± 5 mmol C/m**2/d in spring and 10 ± 2 mmol C/m**2/d in summer. Average e-ratios calculated on a station-by-station basis decreased by ~ 30% from spring to summer, from 0.46 ± 0.48 in ice-covered and MIZ waters, to 0.33 ± 0.26 in summer, though the high uncertainty prevents a statistical differentiation of these data.

Tab. 1+2: Representative electron-microprobe analyses of minerals from Du Toit Nunataks

An example of cordierite-bearing gneiss that is part of a high-grade gneiss-migmatite sequence is described from the Hatch Plain in the Read Mountains of the Shackleton Range, Antarctica, for the first time. The cordierite-bearing rocks constitute the more melanosomic portions of the metatectic and migmatitic rocks that are associated with relict granulite facies rocks such as enderbitic granulite and enderbitic garnet granulite. The predominant mineral assemblage in the cordierite-bearing rocks is chemically homogeneous cordierite (XMg 0.61) and biotite (XMg 0.47), strongly zoned garnet (XMg 0.18-0.11), sillimanite, K-feldspar (Or81-94Ab5-18An0.6), plagioclase (An28), and quartz. Inclusions of sillimanite and biotite relics in both garnet and cordierite indicate that garnet and cordierite were produced by the coupled, discontinuous reaction biotite + sillimanite + quartz = cordierite + garnet + K-feldspar + H2O. Various garnet-biotite and garnet-cordierite geothermometers and sillimanite-quartz-plagioclase-garnet-cordierite geobarometers yield a continuous clockwise path in the P-T diagram. The P-T conditions for equilibrium between garnet core and cordierite and between garnet core and biotite during peak metamorphism and migmatization were estimated to be 690 °C at 5-6 kb. This was followed by cooling and unloading with continuously changing conditions down to 515 °C at 2-3 kb. This low-pressure re-equilibration correlates with the pressure conditions evaluated by SCHULZE (1989) for the widespread granitic gneisses of the Read Group in the Shackleton Range. The associated relict enderbitic granulites representing low-pressure type granulite (8 kb; 790 °C) are comparable to similar low-pressure granulites from the East Antarctic craton. They were either formed by under-accretion processes after collision (WELLS 1979, p. 217) or they are a product of remetamorphism at P-T conditions intermediate between granulite and amphibolite facies. A model of a multiple imbrication zone with crustal thickening (CUTHBERT et al. 1983) is discussed for the formation of the relict granulites of the central and eastern Read Mountains which show higher pressure conditions (8-12 kb, SCHULZE & OLESCH 1990), indicating a Proterozoic crustal thickness of at least 40 km.

(Table 2) Vertical fluxes of organic carbon and sediment matter from the photic zone in the Arctic Basin

On the basis of analysis of satellite and field data collected in Russian Arctic Seas maps of distribution of primary production for different months of the vegetation period were compiled. These maps were used to estimate annual primary production of organic carbon: 55 million tons in the Barents Sea; about 20 million tons in the Kara Sea; 10-15 million tons in the Laptev Sea and in the East Siberian Sea, 42 million tons in the Chukchi Sea. In the central and eastern parts of the Barents Sea during the vegetation period values of primary production decreased by factor >5 (from >500 to <100 mg C/m**2/day). By reviewing results of studies with sediment traps vertical fluxes of organic carbon in different regions of the Arctic Basin were estimated. Significant temporal variability of Corg fluxes with maxima during phytoplankton blooms (by 830 mg C/m**2/day) was noted. Typical summer fluxes of Corg are 10-40 mg C/m**2/day in the southern Barents Sea, 1-10 mg C/m**2/day in the northern Barents Sea and in the Kara Sea, and up to 370 mg C/m**2/day in the zone of marginal filters of the Ob and Yenisey rivers.

Characteristics of eggs and female adults of Calanus finmarchicus, C. glacialis and C. hyperboreus from Disko Bay, western Greenland

This is the first high temporal-resolution study in Disko Bay covering population dynamics, grazing, reproduction, and biochemical composition of 3 dominating copepod species (Calanus finmarchicus, C. glacialis and C. hyperboreus) from late winter to midsummer in 2008. C. finmarchicus and C. glacialis ascended to the surface layer at the onset of the spring phytoplankton bloom, followed by C. hyperboreus 2 wk later. C. finmarchicus spawning occurred during the bloom and postbloom period, partially fueled by wax esters. C. glacialis commenced spawning before the bloom, yet it was greatly stimulated when food became available. However, feeding and reproduction was terminated after the main bloom despite the presence of food. In terms of feeding, this was also the strategy for C. hyperboreus. Between pre-bloom and post-bloom, C. finmarchicus showed an increase in carbon, nitrogen, and phospholipid content but a decrease in total lipid content. This was likely the result of protein synthesis, oocyte maturation, and spawning fueled by wax esters and by feeding. C. glacialis showed a similar pattern, although with an increasing total lipid content from pre-bloom to post-bloom, and an increasing wax ester and decreasing phospholipid content after reproduction was terminated. C. hyperboreus showed greatly increased content of carbon, nitrogen, and all lipid classes between the pre- and post-bloom periods. Hence, C. finmarchicus commenced feeding and spawning at the onset of the bloom and continued throughout the remaining study period. Both C. glacialis and C. hyperboreus females refueled their storage lipids (wax esters) during the bloom and post-bloom period, suggesting that they may spawn in an additional year.

(Table 2) Production characteristics of phytoplankton and selected related factors at bottle sampling stations in the subtropical and tropical Atlantic Ocean

In October and November 2002, high and relatively high values of chlorophyll a concentration at the sea surface (Cchl) were observed in the English Channel (0.47 mg/m**3), in waters of the North Atlantic Current (0.25 mg/m**3 ), in the tropical and subtropical anticyclonic gyres (0.07-0.42 mg/m**3), and also in the southwestern region of the southern subtropical anticyclonic gyre (usually 0.11-0.23 mg/m**3). The central regions of the southern subtropical anticyclonic gyre (SATG) and the North Atlantic tropical gyre (NATR) were characterized by lower values of Cchl (0.02-0.08 mg/m**3 for the SATG and 0.07-0.14 mg/m**3 for the NATR). At most of the SATG stations, values of surface primary production (Cphs) varied from 2.5 to 5.5 mg C/m**3 per day and were mainly defined by fluctuations of Cchl (r = +0.78) rather than by those of the assimilation number (r = +0.54). Low assimilation activity of phytoplankton in these waters (1.3-4.6 mg chl a per hour) pointed to a lack of nutrients. Analysis of variability of their concentration and composition of photosynthetic pigments showed that, in waters north of 30°N, the growth of phytoplankton was mostly restricted by deficiency of nitrogen, while, in more southern areas, at the majority of stations (about 60%), phosphorus concentrations were minimal. At low concentrations of nitrates and nitrites, ammonium represented itself as a buffer that prevented planktonic algae from extreme degrees of nitric starvation. In tropical waters and in waters of the SATG, primary production throughout the water column varied from 240 to 380 mg C/m**2 30° per day. This level of productivity at stations with low values of C chl (<0.08 mg/m**3) was provided by a well-developed deep chlorophyll maximum and high transparency of water. Light curves of photosynthesis based on in situ measurements point to high efficiency of utilizing penetrating solar radiation by phytoplankton on cloudy days.

(Table 2) Production parameters of phytoplankton and some associated parameters in the area of the Titanic Polygon

Studies were carried out mostly in the area of RMS Titanic wreck site (41°44'N, 49°57'W) located above the continental slope and the south of the Grand Banks of Newfoundland. In a period from 18.06 to 24.09.2001 five surveys of production characteristics of surface phytoplankton were conducted over 5-9 days. Mean values of these characteristics obtained during the surveys were 9.2-11.7 mg C/m**3 per day for primary production (C_phs), 0.102-0.188 mg/m**3 for chlorophyll a (C_chls), and 4.44-7.42 mg C/mg chl. a per hour for assimilation number (AN). The main reason for low C_phs variability was a significant inverse relationship (R=-0.66) between AN and C_chls found over the research area. When cold shelf waters dominated in the area (27.07 to 19.08.2001), C_chls values for the slope region (0.125+/-0.031 µg/l) and for the outer shelf (0.130+/-0.040 µg/l) were similar. During strengthening of influence of warmer slope waters within area (from 29.08 to 13.09.2001), C_chls concentration within surface waters of the outer shelf was 0.152+/-0.039 µg/l and exceeded one for the slope region (0.094+/-0.004 µg/l) by factor 1.6. Against the background of low Cchls values, the High values of integral primary production in the water column (510-1010 mg C/m**2 per day) at low C_chls values measured within the area were determined both by high assimilation activity of phytoplankton and by the deep (30-40 m) maximum of primary production. Main reasons for formation of such a maximum were high chlorophyll concentration within the layer of the deep chlorophyll maximum (up to 0.5-2.5 µg/l) and in the relatively high solar irradiance within this layer varying from 1.4 to 8.6% of subsurface PAR.

Analysis of Manganese nodules from Sonne cruises SO06/1 and /2, "Hawaii-Tahiti-Transect", Teilprojekt ICIME

Manganese nodules research has focused on the area between the Clarion Fracture Zone to the North and the Clipperton Fracture Zone to the South where significant concentrations were found ni Ni-Cu. During the CCOP/SOPAC-IOC/IDOE International workshop on the "Geology Mineral Resources and Geophysics of the South Pacific" held in Fiji in September 1975, a working group on manganese nodules was formed by scientists from: CNEXO, Brest, the Institute of Oceanography, New Zealand, Imperial College, London and the Technical University of Aachen. A draft project was presented in July 1976 by J. Andrews, University of Hawaii and G. Pautot, Cnexo on a joint survey under the name of: "Hawaii-Tahiti Transect program". Further details were worked on in September 1976 during the International Geological Congress in Sydney with the participation of D. Cronan, Imperial College, Glasby, New Zealand Geological Survey and G. Friedrich, Aachen TU. The scientific final program was established in July 1977, planning on the participation of three research vessels: the Suroit (CNEXO), the Kana Keoki (U. of Hawaii) and the Sonne (Aachen TU). Several survey areas were selected across the Pacific Ocean (Areas A, B, C, D, E, F, G and H) with about the same crustal age (about 40 million years) and a similar water depths. Being near large fault zones, the ares would be adequate to study the influences of biological productivity, sedimentation rate and possibly volcanic activity on the formation and growth of manganese nodules. The influnece of volcanic activity study would particularly apply to area G being situated near the Marquesas Fracture Zone. The cruise from R/V Sonne started in August 1978 over areas C, D, F, G K. The R/V suroit conducted a similar expedition in 1979 over areas A, B, C, D, E, H and I. Others cruises were planned during the 1979-1980 for the R/V Kana Keoki. The present text relates the R/V Sonne Cruises SO-06/1 and SO-06/2 held within the frame work of this international cooperative project.

Identification of a homolog of Arabidopsis DSP4 (SEX4) in chestnut: its induction and accumulation in stem amyloplasts during winter or in response to the cold_

Oligosaccharide synthesis is an important cryoprotection strategy used by woody plants during winter dormancy. At the onset of autumn, starch stored in the stem and buds is broken down in response to the shorter days and lower temperatures resulting in the buildup of oligosaccharides. Given that the enzyme DSP4 is necessary for diurnal starch degradation in Arabidopsis leaves, this study was designed to address the role of DSP4 in this seasonal process in Castanea sativa Mill. The expression pattern of the CsDSP4 gene in cells of the chestnut stem was found to parallel starch catabolism. In this organ, DSP4 protein levels started to rise at the start of autumn and elevated levels persisted until the onset of spring. In addition, exposure of chestnut plantlets to 4 °C induced the expression of the CsDSP4 gene. In dormant trees or cold-stressed plantlets, the CsDSP4 protein was immunolocalized both in the amyloplast stroma and nucleus of stem cells, whereas in the conditions of vegetative growth, immunofluorescence was only detected in the nucleus. The studies indicate a potential role for DSP4 in starch degradation and cold acclimation following low temperature exposure during activity–dormancy transition.

A “REVOLTA” DE JEÚ E A ESTELA DE DÃ: Um Estudo Em 2 Reis 10,28-36

A pesquisa tem por objetivo trabalhar o evento da Revolta de Jeú, em conjunto com a Estela de Dã, tendo como ponto de partida para tal, a exegese da perícope de 2 Reis 10-28,36. A história Deuteronomista apresenta o ato da Revolta de Jeú como sendo um feito demasiadamente importante, na restauração do culto a Javé em Israel, a partir de um contexto onde o culto a outras divindades, em Israel Norte, estava em pleno curso. No entanto, a partir da análise conjunta da Estela de Dã, que tem como provável autor o rei Hazael de Damasco, somos desafiados a ler esta história pelas entrelinhas não contempladas pelo texto, que apontam para uma participação ativa de Hazael, nos desfechos referentes a Revolta de Jeú, como sendo o responsável direto que proporcionou a subida de Jeú ao trono em Israel, clarificando desta forma este importante período na história Bíblica. Para tal análise, observar-se-á três distintos tópicos, ligados diretamente ao tema proposto: (1) A Revolta de Jeú e a Redação Deuteronomista, a partir do estudo exegético da perícope de 2 Reis 10,28-36, onde estão descritas informações pontuais sobre período em que Jeú reinou em Israel; (2) Jeú e a Estela de Dã, a partir da apresentação e análise do conteúdo da Estela de Dã, tratando diretamente dos desdobramentos da guerra em Ramote de Gileade, de onde se dá o ponto de partida à Revolta de Jeú; e por fim (3) O Império da Síria, onde a partir da continuidade da análise do conteúdo da Estela de Dã, demonstraremos a significância deste reino, além de apontamentos diretamente ligados ao reinado de Hazael, personagem mui relevante no evento da Revolta de Jeú.

Macrophage inflammatory protein-2: Chromosomal regulation in rat small intestinal epithelial cells

Nonpathogenic, resident bacteria participate in the pathogenesis of inflammation in the small intestine, but the molecular messages produced by such bacteria are unknown. Inflammatory responses involve the recruitment of specific leukocyte subsets. We, therefore, hypothesized that butyrate, a normal bacterial metabolite, may modulate chemokine secretion by epithelial cells, by amplifying their response to proinflammatory signals. We studied the expression of the chemokine, macrophage inflammatory protein-2 (MIP-2) by the rat small intestinal epithelial cell line, IEC-6. Cells were stimulated with lipopolysaccharide or with interleukin 1β (IL-1β) and incubated with sodium butyrate. Acetylation of histones was examined in Triton X acetic acid–urea gels by PAGE. Unstimulated IEC-6 cells did not secrete MIP-2. However, lipopolysaccharide and IL-1β induced MIP-2 expression. Butyrate enhanced MIP-2 secretion both in lipopolysaccharide-stimulated and IL-1β-stimulated enterocytes; but butyrate alone did not induce MIP-2 expression. Butyrate increased the acetylation of histones extracted from the nuclei of IEC-6 cells. Furthermore, acetylation of histones (induced by trichostatin A, a specific inhibitor of histone deacetylase) enhanced MIP-2 expression by cells stimulated with IL-1β. In conclusion, trichostatin A reproduced the effects of butyrate on MIP-2 secretion. Butyrate, therefore, increases MIP-2 secretion in stimulated cells by increasing histone acetylation. We speculate that butyrate carries information from bacteria to epithelial cells. Epithelial cells transduce this signal through histone deacetylase, modulating the secretion of chemokines.

The neuronal RNA-binding protein Nova-2 is implicated as the autoantigen targeted in POMA patients with dementia

Paraneoplastic opsoclonus myoclonus ataxia (POMA) is a neurologic disorder thought to be mediated by an autoimmune attack against onconeural disease antigens that are expressed by gynecologic or lung tumors and by neurons. One POMA disease antigen, termed Nova-1, has been identified as a neuron-specific KH-type RNA-binding protein. Nova-1 expression is restricted to specific regions of the central nervous system, primarily the hindbrain and ventral spinal cord, which correlate with the predominantly motor symptoms in POMA. However, POMA antisera recognize antigens that are widely expressed in both caudal and rostral regions of the central nervous system, and some patients develop cognitive symptoms. We have used POMA antisera to clone a cDNA encoding a second POMA disease antigen termed Nova-2. Nova-2 is closely related to Nova-1, and is expressed at high levels in neurons during development and in adulthood, and at lower levels in the adult lung. In the postnatal mouse brain, Nova-2 is expressed in a pattern that is largely reciprocal with Nova-1, including high levels of Nova-2 expression in the neocortex and hippocampus. Functional characterization of Nova-2 in RNA selection and nitrocellulose filter-binding assays reveals that Nova-2 binds RNA with high affinity and with sequence specificity that differs from Nova-1. Our results demonstrate that the immune response in POMA targets a family of highly related sequence-specific neuronal RNA-binding proteins. The expression pattern of the Nova-2 protein is likely to underlie the development of cognitive deficits in some POMA patients.

Characterization and tissue-specific expression of the rat basic fibroblast growth factor antisense mRNA and protein

An RNA transcribed from the antisense strand of the FGF-2 gene has been implicated in the regulation of FGF-2 mRNA stability in amphibian oocytes. We have now cloned and characterized a novel 1.1-kb mRNA (fgf-as) from neonatal rat liver. In non-central nervous system (CNS) tissues the fgf-as RNA is abundantly expressed in a developmentally regulated manner. The FGF-AS cDNA contains a consensus polyadenylylation signal and a long open reading frame (ORF) whose deduced amino acid sequence predicts a 35-kDa protein with homology to the MutT family of nucleotide hydrolases. Western blot analysis with antibodies against the deduced peptide sequence demonstrates that the FGF-AS protein is expressed in a broad range of non-CNS tissue in the postnatal period. In the developing brain, the abundance of sense and antisense transcripts are inversely related, suggesting a role for the antisense RNA in posttranscriptional regulation of FGF-2 expression in this tissue.The FGF-AS is complementary to two widely separated regions in the long 3′ untranslated region of the FGF-2 mRNA, in the vicinity of the proximal and distal polyadenylylation sites. These findings demonstrate that the FGF-2 and fgf-as RNAs are coordinately transcribed on a tissue-specific and developmentally regulated basis and suggest that interaction of the sense and antisense RNAs may result in posttranscriptional regulation of FGF-2 in some tissues.

Thrombospondin-2: A potent endogenous inhibitor of tumor growth and angiogenesis

Recent evidence suggests a potential role for thrombospondin-2 (TSP-2), a matricellular glycoprotein, in the regulation of primary angiogenesis. To directly examine the biological effect of TSP-2 expression on tumor growth and angiogenesis, human A431 squamous cell carcinoma cells, which do not express TSP-2, were stably transfected with a murine TSP-2 expression vector or with vector alone. A431 cells expressing TSP-2 did not show an altered growth rate, colony-forming ability, or susceptibility to induction of apoptosis in vitro. However, injection of TSP-2-transfected clones into the dermis of nude mice resulted in pronounced inhibition of tumor growth that was significantly stronger than the inhibition observed in A431 clones stably transfected with a thrombospondin-1 (TSP-1) expression vector, and combined overexpression of TSP-1 and TSP-2 completely prevented tumor formation. Extensive areas of necrosis were observed in TSP-2-expressing tumors, and both the density and the size of tumor vessels were significantly reduced, although tumor cell expression of the major tumor angiogenesis factor, vascular endothelial growth factor, was maintained at high levels. These findings establish TSP-2 as a potent endogenous inhibitor of tumor growth and angiogenesis.