NADPH-CYTOCHROME P-450 REDUCTASE: EVIDENCE FOR TWO SEPARATE STRUCTURAL DOMAINS AND ISOLATION AND CHARACTERIZATION OF THE MEMBRANE ASSOCIATED SEGMENT

Homogenous detergent-solubilized NADPH-Cytochrome P-450 reductase was incorporated into microsomes and liposomes. This binding occurred spontaneously at temperatures between 4(DEGREES) and 37(DEGREES) and appeared to involve hydrophobic forces as the binding was not disrupted by 0.5 M sodium chloride. This exogenously-added reductase was active catalytically towards native cytochrome P-450, suggesting an association with the microsomal membrane similar to endogenous reductase. Homogeneous detergent-solubilized reductase was disaggregated by Renex-690 micelles, confirming the presence of a hydrophobic combining region on the enzyme. In contrast to these results, steapsin protease-solubilized reductase was incapable of microsomal attachment and did not interact with Renex-690 micelles. Detergent-solubilized reductase (76,500 daltons) was converted into a form with the electrophoretic mobility of steapsin protease-solubilized reductase (68,000 daltons) and a 12,500 dalton peptide (as determined by polyacrylamide-SDS gel electrophoresis) when the liposomal-incorporated enzyme was incubated with steapsin protease. The 68,000 dalton fragment thus obtained had properties identical with steapsin protease-solubilized reductase, i.e. it was catalytically active towards cytochrome c but inactive towards cytochrome P-450 and did not bind liposomes. The 12,500 dalton fragment remained associated with the liposomes when the digest was fractionated by gel filtration, suggesting that this is the segment of the enzyme which is embedded in the phospholipid bilayer. Thus, detergent-solubilized reductase appears to contain a soluble catalytic domain and a separate and separable membrane-binding domain. This latter domain is required for attaching the enzyme to the membrane and also to facilitate the catalytic interaction between the reductase and its native electron acceptor, cytochrome P-450. The membrane-binding segment of the reductase was isolated by preparative gel electrophoresis in SDS following its generation by proteolytic treatment of liposome-incorporated reductase. The peptide has a molecular weight of 6,400 as determined by gel filtration in 8 M guanidine hydrochloride and has an amino acid composition which is not especially hydrophobic. Following removal of SDS and dialysis out of 6 M urea, the membrane-binding peptide was unable to inhibit the activity of a reconstituted system containing purified reductase and cytochrome P-450. Moreover, when reductase and cytochrome P-450 were added to liposomes which contained the membrane-binding peptide, it was determined that mixed function oxidase activity was reconstituted as effectively as when vesicles without the membrane-binding peptide were used. Thus, the membrane-binding peptide was ineffective as an inhibitor of mixed function oxidase activity, suggesting perhaps that it facilitates catalysis by anchoring the catalytic domain of the reductase proximal to cytochrome P-450 (i.e. in the same mixed micelle) rather than through a specific interaction with cytochrome P-450. ^

AN INVESTIGATION OF THE EFFECT OF FORMALDEHYDE SPECIES ON ENVIRONMENTAL MEASUREMENT METHODS UNDER AMBIENT CONDITIONS

Species variations in formaldehyde solutions and gases were investigated by means of infrared spectral analysis. Double beam infrared spectrometry in conjunction with sodium chloride wafer technique and solvent compensation technique were employed. Formaldehyde species in various solutions were investigated. Formalin 37% was stable for many months. Refrigeration had no effects on its stability. Spectral changes were detected in 1000 ppm formaldehyde solutions. The absorbances of very diluted solutions up to 100 ppm were lower than the detection limit of the instruments. Solvent compensation improved resolution, but was associated with an observed lack of repeatability. Formaldehyde species in animal chambers containing animals and in mobile home air were analyzed with the infrared spectrophotometer equipped with a 10 cm gas cell. Spectra were not different from the spectrum of clean air. A portable single beam infrared spectrometer with a 20 meter pathlength was used for reinvestigation. Indoor formaldehyde could not be detected in the spectral; conversely, an absorption peak at 3.58 microns was found in the spectra of 3 and 15 ppm formaldehyde gas in animal chambers. This peak did not appear in the spectrum of the control chamber. Because of concerns over measurement bias among various analytical methods for formaldehyde, side-by-side comparisons were conducted in both laboratory and field measurements. The chromotropic acid method with water and 1% sodium bisulfite as collection media, the pararosaniline method, and a single beam infrared spectrometer were compared. Measurement bias was elucidated and the extent of the effects of temperature and humidity was also determined. The problems associated with related methods were discussed. ^

Molecular interactions of the central conserved domain of p53

p53 mutations are the most commonly observed genetic alterations in human cancers to date. A majority of these point mutations cluster in four evolutionarily conserved domains spanning amino acids 100-300. This region of p53 has been called its central conserved, or conformational domain. This domain of p53 is also targeted by the SV40 T antigen. Mutation, as well as interaction with SV40 T antigen results in inactivation of p53. We hypothesized that mutations and SV40 T antigen disrupt p53 function by interfering with the molecular interactions of the central conserved domain. Using a chimeric protein consisting of the central conserved domain of wild-type p53 (amino acids 115-295) and a protein A affinity tail, we isolated several cellular proteins that interact specifically with this domain of p53. These proteins range in size from 30K to 90K M$\rm\sb{r}.$ We also employed the p53 fusion protein to demonstrate that the central conserved domain of p53 possesses sequence-specific DNA-binding activity. Interestingly, the cellular proteins binding to the central conserved domain of p53 enhance the sequence-specific DNA-binding activity of full length p53. Partial purification of the individual proteins binding to the conformational domain of p53 by utilizing a sodium chloride step-gradient enabled further characterization of two proteins: (1) a 42K M$\rm\sb{r}$ protein that eluted at 0.5M NaCl, and bound DNA nonspecifically, and (2) a 35K M$\rm\sb{r}$ protein eluting into the 1.0M NaCl fraction, capable of enhancing the sequence-specific DNA-binding activity of p53. In order to determine the physiologic relevance of the molecular interactions of the conformational domain of p53, we examined the biochemical processes underlying the TNF-$\alpha$ mediated growth suppression of the NSCLC cell line H460. While growth suppression was accompanied by enhanced sequence-specific p53-DNA binding activity in TNF-$\alpha$ treated H460 nuclei, there was no increase in p53 protein levels. Furthermore, p35 was upregulated in TNF-$\alpha$ treated H460 cells, suggesting that the enhanced p53-DNA binding seen in these cells may be mediated by p35. Our studies define two novel interactions involving the central conserved domain of p53 that appear to be functionally relevant: (1) sequence-specific DNA-binding, and (2) interaction with other cellular proteins. ^

Geochemistry of manganese nodules from a British Columbia fjord

Concretions of iron and manganese oxides and hydrous oxidesóobjects commonly called manganese nodulesóare widely distributed not only on the deep-sea floor but also in shallow marine environments1. Such concretions were not known to occur north of Cape Mendocino in the shallow water zones bordering the North-East Pacific Ocean until the summer of 1966 when they were recovered by one of us (J. W. M.) in dredge samples from Jervis Inlet, a fjord approximately 50 miles north-west of Vancouver, British Columbia.

Medallones de carne vacuna : reducción del contenido de sal por incorporación de oleorresinas de especias

Para evaluar la posible disminución de la adición de cloruro de sodio, mediante el agregado de oleorresinas de especias, se elaboró una masa en la que se empleó 85 % de carne vacuna y 15 % de grasa. Se fraccionó en tres lotes, adicionándoles: 1000 mg/kg de oleorresina de Origanum x Majoricum, 750 mg/kg de la de Capsicum annum o de la de Acantholippia seriphioides, respectivamente. Luego se dividieron en cinco porciones iguales y se agregó sal, hasta alcanzar contenidos de 0.00 %; 0.25 %; 0.50 %; 0.75 % y 1.00 %. Se homogeneizaron y se elaboraron medallones de 100 g, que se cocinaron en horno hasta alcanzar una temperatura interna de 72 °C. Se realizó una evaluación sensorial con 15 jueces semientrenados, solicitándoles que asignaran puntajes, mediante escalas estructuradas de siete puntos e indicaran cuál/es rechazarían. Los puntajes asignados por el panel se sometieron a un análisis exploratorio de datos, a las pruebas de Page y de comparaciones múltiples. Los medallones adicionados con los tres tipos de oleorresinas para las dosis de sal ensayadas presentaron diferencias (= 0.05): las dosis de 0 y 1% fueron las menos aceptadas y la de 0.25 % fue la más aceptada. El 50 % de los jueces rechazó el medallón con 0 % de sal, para los tres tipos de oleorresinas. En las condiciones ensayadas, la incorporación de oleorresinas en dosis de 1000 mg/kg para orégano o de 750 mg/kg para pimiento y tomillo mendocino, permite formular medallones de carne vacuna, con bajo contenido de sal y alta aceptación.

Humatos de vermicompost como mitigador de la salinidad en albahaca (Ocimum basilicum L.)

Los bioestimulantes del crecimiento son sustancias que actúan como activadores fisiológicos, absorción de nutrientes y mitigan el estrés salino. El objetivo fue evaluar humatos de vermicompost como atenuantes del efecto de la salinidad en la emergencia y crecimiento de albahaca (Ocimum basilicum L.). El diseño experimental fue completamente al azar con arreglo factorial, cuyos factores fueron variedades de albahaca (Napoletano y Sweet Genovese), concentraciones de NaCl (0, 50 y 100 mM) y humatos de vermicompost (0 y 1/60 v/v) con seis repeticiones. Se realizaron análisis de varianza y comparaciones independientes de medias (p ≤ 0,05). El experimento se realizó en 2013 en La Paz, México. Se midió el porcentaje y tasa de emergencia, longitud de radícula, altura de plántula, biomasa fresca y seca de radícula y de parte aérea. Se encontraron diferencias significativas para todas las variables (p ≤ 0 ,05), mostrando respuesta diferencial entre variedades para tasa, porcentaje de emergencia y variables morfométricas, destacando la variedad Napoletano con aplicación del bioestimulante como el tratamiento con mejores resultados. El uso de humatos estimuló todas las variables en condiciones de salinidad, permitiendo que la variedad tolerante mejore su emergencia y crecimiento y la variedad sensible incremente su tolerancia al estrés salino.

Ion concentration and deduced chemical compounds of the GRIP and Dome Fuji ice cores, Greenland and Antarctica

We have proposed a method of deducing the chemical compounds found in deep polar ice cores by analyzing the balance between six major ions (Cl-, NO3-, SO4**2-, Na+, Mg2+, and Ca2+). The method is demonstrated for the Holocene and last glacial maximum regions of the Dome Fuji and GRIP ice cores. The dominant compounds depend only on the ion balance and the sequence of chemical reactions. In priority order, the principle salts are calcium sulfate, other sulfates, nitrate, chloride, and carbonate. The chemical abundances deduced by this method agree well with the results of Raman spectroscopy on individual salt inclusions. The abundances in the ice cores are shown to reflect differences in climatic periods (the acidic environment of the Holocene versus the reductive environment of the last glacial maximum) and regional conditions (the marine environment of Antarctica versus the continental environment of Greenland).

Geochemistry and mineralogy of Eocene-Oligocene sediments of IODP Hole 302-M0002A

In 2004, Integrated Ocean Drilling Program Expedition 302 (Arctic Coring Expedition, ACEX) to the Lomonosov Ridge drilled the first Central Arctic Ocean sediment record reaching the uppermost Cretaceous (~430 m composite depth). While the Neogene part of the record is characterized by grayish-yellowish siliciclastic material, the Paleogene part is dominated by biosiliceous black shale-type sediments. The lithological transition between Paleogene and Neogene deposits was initially interpreted as a single sedimentological unconformity (hiatus) of ~26 Ma duration, separating Eocene from Miocene strata. More recently, however, continuous sedimentation on Lomonosov Ridge throughout the Cenozoic was proclaimed, questioning the existence of a hiatus. In this context, we studied the elemental and mineralogical sediment composition around the Paleogene-Neogene transition at high resolution to reconstruct variations in the depositional regime (e.g. wave/current activity, detrital provenance, and bottom water redox conditions). Already below the hiatus, mineralogical and geochemical proxies imply drastic changes in sediment provenance and/or weathering intensity in the hinterland, and point to the existence of another, earlier gap in the sediment record. The sediments directly overlying the hiatus (the Zebra interval) are characterized by pronounced and abrupt compositional changes that suggest repeated erosion and re-deposition of material. Regarding redox conditions, euxinic bottom waters prevailed at the Eocene Lomonosov Ridge, and became even more severe directly before the hiatus. With detrital sedimentation rates decreasing, authigenic trace metals were highly enriched in the sediment. This continuous authigenic trace metal enrichment under persistent euxinia implies that the Arctic trace metal pool was renewed continuously by water mass exchange with the world ocean, so the Eocene Arctic Ocean was not fully restricted. Above the hiatus, extreme positive Ce anomalies are clear signs of a periodically well-oxygenated water column, but redox conditions were highly variable during deposition of the Zebra interval. Significant Mn enrichments only occur above the Zebra interval, documenting the Miocene establishment of stable oxic conditions in the Arctic Ocean. In summary, extreme and abrupt changes in geochemistry and mineralogy across the studied sediment section do not suggest continuous sedimentation at the Lomonosov Ridge around the Eocene-Miocene transition, but imply repeated periods of very low sedimentation rates and/or erosion.

First results of SONNE cruise SO121

The Red Sea is a very young ocean, and is one of the most interesting areas on Earth (ocean in statu nascendi). It is the only ocean where hydrothermal activity associated with ore formation occurs in a sterile environment (anoxic, hot, saline). In addition, its geographical position means that it is predestined to record the monsoonal history of the region in detailed sedimentary sequences. The major aim of the present project is to investigate the dynamics of hydrothermal systems in selected Deeps (Atlantis-II, Discovery, Kebrit, Al Wajh), Additional palaeoceanographic and microbiological questions should also be addressed. Specific aims are: 1. To study the hydrographic changes in individual Deeps (hydrothermal region Atlantis-II) and to investigate the causes of the temperature increase in the last few years (increased heat flow - higher temperature of the brine supply - higher brine flow rates?). 2.a. To document the influence of the hydrothermal systems on the sedimentary organic matter in the Deeps. In particular, the thermogenic production and migration of hydrocarbons in the sediments will be studied. The complex formation mechanisms (bacterial, thermogenic) of short-chain hydrocarbons (trace gases) will also be examined, 2.b. in addition, the polar and macromolecular fraction in samples from the various deeps will be studied in order to elucidate the formation, structure and source of the macromolecular oil fraction. 3. To clarify the palaeoceanographic conditions, sea-level changes and the climatic history (relationship of the circulation system and nutrient supply to the monsoon) of the southern Red Sea. 4. To separate microorganisms from the brines and to characterise them in terms of their metabolic physiology and ecology, and to describe their taxonomy.

Results of bulk sediment X-ray diffraction analysis and quantification of mineral phases based on the RockJock and on the QUAX quantitative analysis

We have performed quantitative X-ray diffraction (qXRD) analysis of 157 grab or core-top samples from the western Nordic Seas between (WNS) ~57°-75°N and 5° to 45° W. The RockJock Vs6 analysis includes non-clay (20) and clay (10) mineral species in the <2 mm size fraction that sum to 100 weight %. The data matrix was reduced to 9 and 6 variables respectively by excluding minerals with low weight% and by grouping into larger groups, such as the alkali and plagioclase feldspars. Because of its potential dual origins calcite was placed outside of the sum. We initially hypothesized that a combination of regional bedrock outcrops and transport associated with drift-ice, meltwater plumes, and bottom currents would result in 6 clusters defined by "similar" mineral compositions. The hypothesis was tested by use of a fuzzy k-mean clustering algorithm and key minerals were identified by step-wise Discriminant Function Analysis. Key minerals in defining the clusters include quartz, pyroxene, muscovite, and amphibole. With 5 clusters, 87.5% of the observations are correctly classified. The geographic distributions of the five k-mean clusters compares reasonably well with the original hypothesis. The close spatial relationship between bedrock geology and discrete cluster membership stresses the importance of this variable at both the WNS-scale and at a more local scale in NE Greenland.

Mineral quantification of a Jordan Cretaceous outcrop (Section GM3, Ghawr Al Mazar, Ghor al Mazrar)

Cyclic fluctuations in global sea level during epochs of warm greenhouse climate have remained enigmatic, because absence or subordinate presence of polar ice during these periods precludes an explanation by glacio-eustatic forcing. An alternative concept suggests that the water-bearing potential of groundwater aquifers is equal to that of ice caps and that changes in the dynamic balance of aquifer charge versus discharge, as a function of the temperature-related intensity of the hydrological cycle, may have driven eustasy during warm climates. However, this idea has long been neglected for two reasons: 1) the large storage potential of subsurface aquifers was confused with the much smaller capacity of rivers and lakes and 2) empirical data were missing that document past variations in the hydrological cycle in relation to eustasy. In the present study we present the first empirical evidence for changes in precipitation, continental weathering intensity and evaporation that correlate with astronomically (long obliquity) forced sea-level cycles during the warmest period of the Cretaceous (Cenomanian-Turonian). We compare sequence-stratigraphic data with changes in the terrigenous mineral assemblage in a low-latitude marine sedimentary sequence from the equatorial humid belt at the South-Tethyan margin (Levant carbonate platform, Jordan), thereby avoiding uncertainties from land-ocean correlations. Our data indicate covariance between cycles in weathering and sea level: predominantly chemical weathering under wet climate conditions is reflected by dominance of weathering products (clays) in deposits that represent sea-level fall (aquifer charge > discharge). Conversely, preservation of weathering-sensitive minerals (feldspars, epidote and pyroxenes) in transgressive sediments reflects decreased continental weathering due to dryer climate (aquifer discharge > charge). Based on our results we suggest that aquifer-eustasy represents a viable alternative to glacio-eustasy as a driver of cyclic 3rd-order sea-level fluctuations during the middle Cretaceous greenhouse climate, and it may have been a pervasive process throughout Earth history.