Inactivation and self -association of CaM kinase: Effects of ATP, substrate binding domains, and isozymic forms

Calcium/calmodulin-dependent protein kinase II (CaM kinase) is a multifunctional Ser/Thr protein kinase, that is highly enriched in brain and is involved in regulating many aspects of neuronal function. We observed that forebrain CaM kinase from crude homogenates, cytosolic fractions and purified preparations inactivates and translocates into the particulate fraction following autophosphorylation. Using purified forebrain CaM kinase as well as recombinant $\alpha$ isozyme, we determined that the formation of particulate enzyme was due to enzyme self-association. The conditions of autophosphorylation determine whether enzyme self-association and/or inactivation will occur. Self-association of CaM kinase is sensitive to pH, ATP concentration, and enzyme autophosphorylation. This process is prevented by saturating concentrations of ATP. However, in limiting ATP, pH is the dominant factor, and enzyme self-association occurs at pH values $\rm{<}7.0.$ Site-specific mutants were produced by substituting Ala for Thr286, Thr253, or Thr305,306 to determine whether these sites of autophosphorylation affect enzyme inactivation and self-association. The only mutation that influenced these processes was Ala286, which removed the protective effect afforded by autophosphorylation in saturating ATP. Enzyme inactivation occurs in the presence and absence of self-association and appears predominantly sensitive to nucleotide concentration, because saturating concentrations of $\rm Mg\sp{2+}/ADP$ or $\rm Mg\sp{2+}/ATP$ prevent this process. These data implicate the ATP binding pocket in both inactivation and self-association. We also observed that select peptide substrates and peptide inhibitors modeled after the autoregulatory domain of CaM kinase prevented these processes. The $\alpha$ and $\beta$ isozymes of CaM kinase were characterized independently, and were observed to exhibit differences in both enzyme inactivation and self-association. The $\beta$ isozyme was less sensitive to inactivation, and was never observed to self-associate. Biophysical characterization, and transmission electron microscopy coupled with image analysis indicated both isozymes were multimeric, however, the $\alpha$ and $\beta$ isozymes appeared structurally different. We hypothesize that the $\alpha$ subunit of CaM kinase plays both a structural and enzymatic role, and the $\beta$ subunit plays an enzymatic role. The ramifications for the functional differences observed for inactivation and self-association are discussed based on potential structural differences and autoregulation of the $\alpha$ and $\beta$ isozymes in both calcium-induced physiological and pathological processes. ^

Pencil -beam redefinition algorithm dose calculations for electron therapy treatment planning

The electron pencil-beam redefinition algorithm (PBRA) of Shiu and Hogstrom has been developed for use in radiotherapy treatment planning (RTP). Earlier studies of Boyd and Hogstrom showed that the PBRA lacked an adequate incident beam model, that PBRA might require improved electron physics, and that no data existed which allowed adequate assessment of the PBRA-calculated dose accuracy in a heterogeneous medium such as one presented by patient anatomy. The hypothesis of this research was that by addressing the above issues the PBRA-calculated dose would be accurate to within 4% or 2 mm in regions of high dose gradients. A secondary electron source was added to the PBRA to account for collimation-scattered electrons in the incident beam. Parameters of the dual-source model were determined from a minimal data set to allow ease of beam commissioning. Comparisons with measured data showed 3% or better dose accuracy in water within the field for cases where 4% accuracy was not previously achievable. A measured data set was developed that allowed an evaluation of PBRA in regions distal to localized heterogeneities. Geometries in the data set included irregular surfaces and high- and low-density internal heterogeneities. The data was estimated to have 1% precision and 2% agreement with accurate, benchmarked Monte Carlo (MC) code. PBRA electron transport was enhanced by modeling local pencil beam divergence. This required fundamental changes to the mathematics of electron transport (divPBRA). Evaluation of divPBRA with the measured data set showed marginal improvement in dose accuracy when compared to PBRA; however, 4% or 2mm accuracy was not achieved by either PBRA version for all data points. Finally, PBRA was evaluated clinically by comparing PBRA- and MC-calculated dose distributions using site-specific patient RTP data. Results show PBRA did not agree with MC to within 4% or 2mm in a small fraction (<3%) of the irradiated volume. Although the hypothesis of the research was shown to be false, the minor dose inaccuracies should have little or no impact on RTP decisions or patient outcome. Therefore, given ease of beam commissioning, documentation of accuracy, and calculational speed, the PBRA should be considered a practical tool for clinical use. ^

Organic compounds in sediments and pore waters of ODP Sites 117-723 and 117-724

Total organic carbon, amino compounds, and carbohydrates were measured in pore waters and sediments of Pliocene to Pleistocene age from Sites 723 and 724 (ODP Leg 117) to evaluate (1) relationships between organic matter in the sediment and in the pore water, (2) the imprint of lithological variations on the abundance and contribution of organic substances, (3) degradation of amino compounds and carbohydrates with time and/or depth, and (4) the dependence of the ammonia concentration in the pore water on the degradation of amino compounds in the sediment. Total organic carbon concentrations (TOC) of the investigated sediment samples range from 0.9% to 8.7%, and total nitrogen concentrations (TN) from 0.1% to 0.5%. Up to 4.9% of the TOC is contributed by hydrolyzable amino acids (THAA) which are present in amounts between 1.1 and 21.3 µmol/g dry sediment and decrease strongly downhole. Hydrolyzable carbohydrates (THCHO) were found in concentrations from 1.3 to 6.6 ?mol/g sediment constituting between 0.1% and 2.0% of the TOC. Differences between the distribution patterns of monomers in Sites 723 and 724 indicate higher terrigenous influence for Site 724 and, furthermore, enhanced input of organic matter that is relatively resistant to microbial degradation. Lithologically distinct facies close to the Pliocene/Pleistocene boundary yield different organic matter compositions. Laminated horizons seem to correspond with enhanced amounts of biogenic siliceous material and minor microbiological degradation. Total amounts of dissolved organic carbon (DOC) in pore waters vary between 11 and 131 mg/L. Concentrations of DOC as well as of dissolved amino compounds and carbohydrates appear to be related to microbial activity and/or associated redox zones and not so much to the abundance of organic matter in the sediments. Distributions of amino acids and monosaccharides in pore waters show a general enrichment in relatively stable components in comparison to those of the sediments. Nevertheless, the same trend appears between amino acids present in the sediments from Sites 723 and 724 as well as between amino acids in pore waters from these two sites, indicating a direct relation between the dissolved and the sedimentary organic fractions. Different ammonia concentrations in the pore waters of Sites 723 and 724 seem to be related to enhanced release of ammonia from degradation of amino compounds in Site 723.

Geochemistry of pore water and sediments from ODP Leg 136 sites

During Leg 136 drilling was conducted at two sites in pelagic sediments of the north central Pacific Ocean. In this report, pore-water analyses for major seawater constituents, alkalinity, ammonia, nitrate, phosphate, silica, Ba, Fe, Li, Mn, and Sr are presented. Although concentration gradients are generally weak, resulting from slow sedimentation and concomitant diffusive communication with overlying water, there is evidence of sediment/pore-water interactions, associated sediment diagenesis, and formation of authigenic minerals. Bulk major and trace element compositions of the sediments are consistent with reactions inferred to occur within the sediments and with the lithology and mineralogy. Elemental compositions of the sediments are not strongly affected by diagenesis and are primarily related to the dominant mineralogy. Sediments are typical of deep ocean pelagic settings with a significant contribution from the alteration of volcanic ash and the formation of zeolites. Sedimentary rare earth element patterns also provide evidence of active scavenging processes by Mn and Fe oxide phases in the deeper sediments at Site 842.

Stable-isotopes and calcium carbonate at DSDP Hole 73-524

This paper presents a compilation of stable-isotope and percentage-of-carbonate data for the Upper Cretaceous/ lower Tertiary hemipelagic sediments from DSDP Leg 73, Site 524.

Grain size analyses of ODP Leg 178 sites

Sites 1095, 1096, and 1101 were drilled on the continental rise west of the Antarctic Peninsula (Fig. F1) to recover a continuous high-resolution record of Antarctic glaciation. Site 1095 is the subject of a short paper in this volume (Pudsey, 2001, doi:10.2973/odp.proc.sr.178.214.2001), whereas mass accumulation rates at the three sites are described by Wolf-Welling (2001, doi:10.2973/odp.proc.sr.178.223.2001) and ice-rafted debris at Site 1101 is discussed by Cowan (2001, doi:10.2973/odp.proc.sr.178.206.2001) This report documents grain-size data (sand and fine fraction) and the proportion of biogenic silica for the upper 300 m at Site 1095, the upper 250 m at Site 1096, and the whole 220 m at Site 1101.

Organic matter, isotopic composition of calcite veins in basement basalt and pore water at DSDP Leg 78A Holes

Sediments of the Barbados Ridge complex, cored on DSDP Leg 78A, contain low concentrations of acid-insoluble carbon (0.05-0.25%) and nitrogen (C/N 1.5-5) and dispersed C1-C6 hydrocarbons (100-800 ppb). The concentrations of organic carbon and 13C in organic carbon decrease with depth, whereas the concentration of dispersed hydrocarbons increases slightly with depth. These trends may reflect the slow oxidation of organic matter, with selective removal of 13C and slow conversion of the residual organic matter to hydrocarbons. Very minor indications of nitrogen gas were observed at about 250 meters sub-bottom at two of the drilling sites. Basement basalts have calcite veins with d13C values in the range of 2.0 to 3.2 per mil and d18O-SMOW values ranging from 28.5 to +30.6 per mil. Interstitial waters have d18O-SMOW of 0.2 to -3.5 per mil and dD-SMOW of -2 to -15 per mil. The oxygen isotopic composition of the calcite veins in the basement basalts gives estimated equilibrium fractionation temperatures in the range of 11 to 24°C, assuming precipitation from water with d18O-SMOW in the range of +0.1 to -1.0 per mil. This suggests that basalt alteration and precipitation of vein calcite occurred in contact either with warmer Campanian seawater or, later, with pore water, after burial to depths of 200- 300 meters. Pore waters from all three sites are depleted in deuterium and 18O, and dissolved sulfate is enriched in 34S at Sites 541 and 542, but not at Site 543.

Isotopic composition of interstitial waters from ODP Leg 174A sites

An investigation of the isotopic composition of the interstitial waters was conducted at Sites 1071, 1072, and 1073 on the New Jersey continental shelf and slope during Ocean Drilling Program Leg 174A. Sites 1071 and 1072 are closely spaced drill holes on the continental shelf located ~130 km from the shoreline in 88 and 98 m of water, respectively. Site 1073 is located on the continental slope in 640 m water and penetrated a total of 664 m of sediment of which ~520 m is Quaternary age. A total of 125 oxygen and hydrogen isotopic analyses of pore fluids are presented from all three sites. Twelve strontium isotopic ratios are reported from Site 1071.

Age models and foraminifer isotopes of four ODP Leg 202 sites

We present benthic isotope stratigraphies for Sites 1236, 1237, 1239, and 1241 that span the late Miocene-Pliocene time interval from 6 to 2.4 Ma. Orbitally tuned timescales were generated for Sites 1237 and 1241 by correlating the high-frequency variations in gamma ray attenuation density, percent sand of the carbonate fraction, and benthic d13C to variations in Earth's orbital parameters. The astronomical timescales for Sites 1237 and 1241 are in agreement with the one from Atlantic Site 925/926 (Ocean Drilling Program Leg 154). The comparison of benthic d18O and d13C records from the east Pacific sites and Atlantic Site 925/926 revealed a surprising clarity of the "41-k.y. signal" in d13C records and a remarkably good correlation between their d13C records. This suggests that the late Miocene-Pliocene amplitudes of obliquity-related d13C cycles reflect a magnitude of global response often larger than that provided by obliquity-related d18O cycles. At Site 1237, the orbitally derived ages of Pliocene magnetic reversal boundaries between the base of Réunion and the top of Thvera confirm astronomical datings of the generally accepted ATNTS2004 timescale, except for the top of Kaena and the base of Sidufjall. Our astronomical age for the top of Kaena is about one obliquity cycle older. The base of Sidufjall appears to be about one precession cycle younger. The age models of Sites 1236 and 1239 were established by correlating their benthic d18O and d13C records directly to the orbitally tuned isotope record of Site 1241.

Grain-size, carbona and calcium carbonate at DSDP Leg 58 Holes

Sand-silt-clay distribution was determined on 10-cm**3 sediment samples collected at the time the cores were split and described. The sediment classification used here is that of Shepard (1954), with the sand, silt, and clay boundaries based on the Wentworth (1922) scale. Thus the sand, silt, and clay fractions are composed of particles whose diameters are 2000 to 62.5 µm, 62.5 to 3.91 µm, and less than 3.91 µm, respectively. This classification is applied without regard to sediment type and origin; therefore, the sediment names used in this table may differ from those used elsewhere in this volume; e.g., a silt composed of nannofossils may be called a nannofossil ooze in a site chapter.

Late Miocene cyclicity and intercore correlations for ODP Sites 127-794 and 127-797

During Leg 127, the formation microscanner (FMS) logging tool was used as part of an Ocean Drilling Program (ODP) logging program for only the second time in the history of the program. Resistivity images, also known as FMS logs, were obtained at Sites 794 and 797 that covered nearly the complete Yamato Basin sedimentary sequence to a depth below 500 mbsf. The FMS images from these two sites at the northeastern and southwestern corners of the Yamato Basin thus were amenable to comparison. A strong visual correlation was noticed between the FMS logs taken in Holes 794B and 797C in an upper Miocene interval (350-384 mbsf), although the two sites are approximately 360 km apart. In this interval, the FMS logs showed a series of more resistive thin beds (10-200 cm) alternating with relatively lower resistivity layers: a pattern that was manifested by alternating dark (low resistivity) and light (high resistivity) banding in the FMS images. We attribute this layering to interbedding of chert and porcellanite layers, a common lithologic sequence throughout Japan (Tada and Iijima, 1983, doi:10.1306/212F82E7-2B24-11D7-8648000102C1865D). Spatial frequency analysis of this interval of dominant dark-light banding showed spatial cycles of period of 1.1 to 1.3 and 0.6 m. This pronounced layering and the correlation between the two sites terminate at 384 mbsf, coincident with the opal-CT to quartz transition at Site 794. We think the correlation in the FMS logs might well extend earlier in the middle Miocene, but the opal-CT to quartz transition obscures this layering below 384 mbsf. Although 34 m is only a small part of the core recovered at these two sites, it is significant because it represents an area of extremely poor core recovery and an interval for which a near-depositional hiatus was postulated for Site 797, but not for Site 794.

Carbon, hydrocarbon and alkenone composition and indices of ODP Hole 167-1016C sediments

Total organic carbon (TOC) and calcium carbonate (CaCO3) concentrations were determined for 304 samples, and biomarkers were analyzed for 101 samples from Core 167-1016C-1H. TOC varies between 1% and 2%, and CaCO3 is typically 1%-4%, with peaks reaching 14%. Paleotemperature estimated from Uk'37 varies from 8.5° to 17.5°C. The Uk'37 variation implies that Core 167-1016C-1H covers oxygen isotope Stages 1-6. Peaks of diatom-derived C25:1 HBI alkene concentrations occur during warming intervals, suggesting intensified upwelling during deglaciation. The concentrations of haptophyte-derived alkenones and diatom-derived C25:1 HBI alkene vary out of phase, which presumably resulted from the changes in the mode of nutrient supply to surface mixed layer. Maximal CaCO3 contents (>10%) were observed in both warming and cooling intervals. The peak in cooling interval relates to an alkenone maximum, whereas the peaks in warming intervals do not. This implies that carbonate production is not the only factor controlling carbonate compensation depth at this site, and it suggests considering the changes in North Pacific deep-water chemistry. Petroleum-type compounds are present in Site 1016 sediments. Their concentrations are maximized in the warming intervals that correspond to the timing of destruction of a huge tar mound off Point Conception. The tarry material was presumably transported by the Arguello Fan system to Site 1016.

Total number of benthic foraminifera in sediments below the K/T boundary in the Atlantic and Pacific Ocean

Most species of Late Cretaceous deep-sea benthic foraminifera are believed to be cosmopolitan and therefore to exhibit only minor biogeographical differences. In this preliminary report, six Deep Sea Drilling Project (DSDP) sites from different oceans, paleolatitudes, and paleodepths were analyzed for terminal Cretaceous abyssal-bathyal benthic foraminifera in order to investigate their assumed cosmopolitan distribution and the question of whether different faunal compositions are related to time, different paleolatitudes, and/or different paleodepths. The material studied was obtained from the low-latitude Site 465 (Pacific Ocean), and the intermediate-latitude Sites 384 (North Atlantic) and 356, 516, 525, and 527 (South Atlantic). The material analyzed represents a time slice encompassing the last 20-50 k.y. of the Cretaceous. The faunas contain numerous "Velasco-type" species, such as Gavelinella beccariiformis (White), Cibicidoides velascoensis (Cushman), Nuttallides truempyi (Nuttall), Gaudryina pyramidata Cushman, and various gyroidinoids and buliminids. The results contradict the general assumption of the cosmopolitan nature of Late Cretaceous deep-sea benthic foraminifera advocated in the literature. Only about 9% of the taxa identified were found to be truly "cosmopolitan" through their occurrence at all the sites analyzed. On the basis of correspondence analysis and relative abundance data, three assemblages and three subassemblages were recognized: (1) a bathyal-abyssal assemblage [Nuttallinella sp. A, Cibicidoides hyphalus (Fisher), Valvalabamina sp. evolute form, and Gyroidinoides spp.] at the South Atlantic Sites 356, 516, 525, and 527, divided into three subassemblages, namely (a) a middle bathyal subassemblage [Eouvigerina subsculptura McNeil and Caldwell, Truaxia aspera (Cushman), and G. pyramidata] at Sites 516 and 525, (b) a lower bathyal subassemblage [Osangularia? sp., Pyramidina rudita (Cushman and Parker), and Quadrimorphina camerata (Brotzen)] at Site 356, and (c) an abyssal subassemblage [Gyroidinoides sp. C, Hyperammina-Bathysiphon, Gyroidinoides beisseli (White), and Globorotalites sp. B] at Site 527; (2) an abyssal assemblage [Buliminella cf. plana (Cushman and Parker) and Bulimina incisa Cushman] at the North Atlantic Site 384; and (3) a middle bathyal assemblage [Vulvulina sp. A, Osangularia navarroana (Cushman), Alabamina? sp., Bulimina velascoensis (Cushman), Spiroplectammina spp. calcareous forms, and Bulimina trinitatensis Cushman and Jarvis] at the Pacific Site 465.

Pore-water chemistry of ODP Sites 124-767 and 124-768

The combination of multiple sediment sources and varying rates of sediment accumulation in the Celebes and Sulu seas have had significant impact on the processes of diagenesis, mineralization, and pore-fluid flow. Isotopic and mass-balance calculations help elucidate the various reactions taking place in these western Pacific basins, where ash alteration and basalt-seawater interactions are superimposed on the effects of sulfate oxidation of organic carbon and biogenic methane and of dolomitization of biogenic carbonates. Based on the shape of the calcium and magnesium depth profiles, two major reactive zones have been identified. The first is located near the zone of sulfate depletion and is characterized by carbonate recrystallization, dolomitization and ash alteration reactions at both Ocean Drilling Program Sites 767 and 768. The second reactive zone corresponds to the bottom of the sedimentary sequence and is characterized by alteration reactions in the basement (Site 767) and in the pyroclastic deposits beneath the sediment column (Site 768).

Total organic carbon content, methane isotopic composition and hydrocarbon characteristics of ODP Leg 180 sites

The organic geochemistry of Sites 1108 and 1109 of the Woodlark Basin, offshore Papua New Guinea, was studied to determine whether thermally mature hydrocarbons were present in the penetrated section and, if present, whether they are genetically related to the penetrated "coaly" interval. Both the organic carbon and pyrolysis data indicate that there is no significant hydrocarbon source-rock potential at Site 1108. The hydrocarbons encountered during drilling appear to be indigenous and not migrated products or contaminants. In contrast, the coaly interval at Site 1109 contains zones with significant hydrocarbon-generation potential. Several independent lines of evidence indicate that the coaly sequence encountered at Site 1109 is thermally immature. The Site 1108 methane stable-carbon isotope composition does not display a clear trend with depth as would be expected if it was solely reflecting a maturation profile. The measured isotopic composition of methane has most probably been altered by fractionation during sample handling and storage. This fractionation would result in isotopically heavier values than would be obtained on free gas. The organic geochemical data gathered indicate that Site 1108 can be safely revisited and that the organic-rich sediments encountered at Site 1109 were not the source of the gas encountered at Site 1108.