A bait and switch hapten strategy generates catalytic antibodies for phosphodiester hydrolysis

General base catalysis supplied by the histidine-12 (H-12) residue of ribonuclease (RNase) A has long been appreciated as a major component of the catalytic power of the enzyme. In an attempt to harness the catalytic power of a general base into antibody catalysis of phosphodiester bond hydrolysis, the quaternary ammonium phosphate 1 was used as a bait and switch hapten. Based on precedence, it was rationalized that this positively charged hapten could induce a counter-charged residue in the antibody binding site at a locus suitable for it to deprotonate the 2′-hydroxyl group of the anhydroribitol phosphodiester substrate 2. After murine immunization with hapten 1, mAb production yielded a library of 35 antibodies that bound to a BSA-1 conjugate. From this panel, two were found to catalyze the cyclization-cleavage of phosphodiester 2. Kinetic studies at pH 7.49 (Hepes, 20 mM) and 25°C showed that the most active antibody, MATT.F-1, obeyed classical Michaelis–Menten kinetics with a Km = 104 μM, a kcat = 0.44 min−1, and a kcat/kuncat = 1.7 × 103. Hapten 1 stoichiometrically inhibits the catalytic activity of the antibody. MATT.F-1 is the most proficient antibody–catalyst (1.6 × 107 M−1) yet generated for the function of phosphodiester hydrolysis and emphasizes the utility of the bait and switch hapten paradigm when generating antibody catalysts for processes for which general-base catalysis can be exploited.

Energy Sources for HCO3− and CO2 Transport in Air-Grown Cells of Synechococcus UTEX 6251

Light-dependent inorganic C (Ci) transport and accumulation in air-grown cells of Synechococcus UTEX 625 were examined with a mass spectrometer in the presence of inhibitors or artificial electron acceptors of photosynthesis in an attempt to drive CO2 or HCO3− uptake separately by the cyclic or linear electron transport chains. In the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea, the cells were able to accumulate an intracellular Ci pool of 20 mm, even though CO2 fixation was completely inhibited, indicating that cyclic electron flow was involved in the Ci-concentrating mechanism. When 200 μm N,N-dimethyl-p-nitrosoaniline was used to drain electrons from ferredoxin, a similar Ci accumulation was observed, suggesting that linear electron flow could support the transport of Ci. When carbonic anhydrase was not present, initial CO2 uptake was greatly reduced and the extracellular [CO2] eventually increased to a level higher than equilibrium, strongly suggesting that CO2 transport was inhibited and that Ci accumulation was the result of active HCO3− transport. With 3-(3,4-dichlorophenyl)-1,1-dimethylurea-treated cells, Ci transport and accumulation were inhibited by inhibitors of CO2 transport, such as COS and Na2S, whereas Li+, an HCO3−-transport inhibitor, had little effect. In the presence of N,N-dimethyl-p-nitrosoaniline, Ci transport and accumulation were not inhibited by COS and Na2S but were inhibited by Li+. These results suggest that CO2 transport is supported by cyclic electron transport and that HCO3− transport is supported by linear electron transport.

Regulation of Leaf Senescence by Cytokinin, Sugars, and Light : Effects on NADH-Dependent Hydroxypyruvate Reductase

The aim of this study was to investigate the interactions between cytokinin, sugar repression, and light in the senescence-related decline in photosynthetic enzymes of leaves. In transgenic tobacco (Nicotiana tabacum) plants that induce the production of cytokinin in senescing tissue, the age-dependent decline in NADH-dependent hydroxypyruvate reductase (HPR), ribulose-1,5-bisphosphate carboxylase/oxygenase, and other enzymes involved in photosynthetic metabolism was delayed but not prevented. Glucose (Glc) and fructose contents increased with leaf age in wild-type tobacco and, to a greater extent, in transgenic tobacco. To study whether sugar accumulation in senescing leaves can counteract the effect of cytokinin on senescence, discs of wild-type leaves were incubated with Glc and cytokinin solutions. The photorespiratory enzyme HPR declined rapidly in the presence of 20 mm Glc, especially at very low photon flux density. Although HPR protein was increased in the presence of cytokinin, cytokinin did not prevent the Glc-dependent decline. Illumination at moderate photon flux density resulted in the rapid synthesis of HPR and partially prevented the negative effect of Glc. Similar results were obtained for the photosynthetic enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase. It is concluded that sugars, cytokinin, and light interact during senescence by influencing the decline in proteins involved in photosynthetic metabolism.

Temporal fluctuations in coherence of brain waves.

As a measure of dynamical structure, short-term fluctuations of coherence between 0.3 and 100 Hz in the electroencephalogram (EEG) of humans were studied from recordings made by chronic subdural macroelectrodes 5-10 mm apart, on temporal, frontal, and parietal lobes, and from intracranial probes deep in the temporal lobe, including the hippocampus, during sleep, alert, and seizure states. The time series of coherence between adjacent sites calculated every second or less often varies widely in stability over time; sometimes it is stable for half a minute or more. Within 2-min samples, coherence commonly fluctuates by a factor up to 2-3, in all bands, within the time scale of seconds to tens of seconds. The power spectrum of the time series of these fluctuations is broad, extending to 0.02 Hz or slower, and is weighted toward the slower frequencies; little power is faster than 0.5 Hz. Some records show conspicuous swings with a preferred duration of 5-15s, either irregularly or quasirhythmically with a broad peak around 0.1 Hz. Periodicity is not statistically significant in most records. In our sampling, we have not found a consistent difference between lobes of the brain, subdural and depth electrodes, or sleeping and waking states. Seizures generally raise the mean coherence in all frequencies and may reduce the fluctuations by a ceiling effect. The coherence time series of different bands is positively correlated (0.45 overall); significant nonindependence extends for at least two octaves. Coherence fluctuations are quite local; the time series of adjacent electrodes is correlated with that of the nearest neighbor pairs (10 mm) to a coefficient averaging approximately 0.4, falling to approximately 0.2 for neighbors-but-one (20 mm) and to < 0.1 for neighbors-but-two (30 mm). The evidence indicates fine structure in time and space, a dynamic and local determination of this measure of cooperativity. Widely separated frequencies tending to fluctuate together exclude independent oscillators as the general or usual basis of the EEG, although a few rhythms are well known under special conditions. Broad-band events may be the more usual generators. Loci only a few millimeters apart can fluctuate widely in seconds, either in parallel or independently. Scalp EEG coherence cannot be predicted from subdural or deep recordings, or vice versa, and intracortical microelectrodes show still greater coherence fluctuation in space and time. Widely used computations of chaos and dimensionality made upon data from scalp or even subdural or depth electrodes, even when reproducible in successive samples, cannot be considered representative of the brain or the given structure or brain state but only of the scale or view (receptive field) of the electrodes used. Relevant to the evolution of more complex brains, which is an outstanding fact of animal evolution, we believe that measures of cooperativity are likely to be among the dynamic features by which major evolutionary grades of brains differ.

The mode of action of aspirin-like drugs: effect on inducible nitric oxide synthase.

Nitric oxide synthesized by inducible nitric oxide synthase (iNOS) has been implicated as a mediator of inflammation in rheumatic and autoimmune diseases. We report that exposure of lipopolysaccharide-stimulated murine macrophages to therapeutic concentrations of aspirin (IC50 = 3 mM) and hydrocortisone (IC50 = 5 microM) inhibited the expression of iNOS and production of nitrite. In contrast, sodium salicylate (1-3 mM), indomethacin (5-20 microM), and acetaminophen (60-120 microM) had no significant effect on the production of nitrite at pharmacological concentrations. At suprapharmacological concentrations, sodium salicylate (IC50 = 20 mM) significantly inhibited nitrite production. Immunoblot analysis of iNOS expression in the presence of aspirin showed inhibition of iNOS expression (IC50 = 3 mM). Sodium salicylate variably inhibited iNOS expression (0-35%), whereas indomethacin had no effect. Furthermore, there was no significant effect of these nonsteroidal anti-inflammatory drugs on iNOS mRNA expression at pharmacological concentrations. The effect of aspirin was not due to inhibition of cyclooxygenase 2 because both aspirin and indomethacin inhibited prostaglandin E2 synthesis by > 75%. Aspirin and N-acetylimidazole (an effective acetylating agent), but not sodium salicylate or indomethacin, also directly interfered with the catalytic activity of iNOS in cell-free extracts. These studies indicate that the inhibition of iNOS expression and function represents another mechanism of action for aspirin, if not for all aspirin-like drugs. The effects are exerted at the level of translational/posttranslational modification and directly on the catalytic activity of iNOS.

Avaliação do crescimento de Ankistrodesmus braunii em reator tubular empregando diferentes concentrações de nitrato em diferentes condições de cultivo

Microalgas são organismos unicelulares, eucariontes, fotossintetizantes e eficientes fixadores de gás carbônico que apresentam grande potencial para produção de ácidos graxos além de pigmentos, como os carotenóides e a clorofila, de interesse nas indústrias de alimentos, química, farmacêutica e de cosméticos. Dentre as microalgas, o microrganismo Ankistrodesmus braunii vem sendo citado como capaz de produzir grandes quantidades de lipídios, podendo corresponder a até 73% de sua massa seca, com produção de ácidos graxos insaturados, como o ácido linolênico. Esse microrganismo se destaca do ponto de vista industrial por poder ser conduzido em reatores e em meios de cultivo complexos. As fontes de nitrogênio, as concentrações empregadas destes nutrientes, bem como o tipo de processo de cultivo interferem na composição de biomassas fotossintetizantes. O uso de reatores tubulares tem sido estudado e tem se apresentado interessante por permitir a obtenção de altas concentrações celulares. Nesse sentido, este trabalho teve a finalidade de estudar o crescimento de Ankistrodesmus braunii em reator tubular com uso de diferentes quantidades de nitrato de sódio por processos descontínuo, descontínuo alimentado e semi-contínuo. Nos cultivos descontínuos, a máxima concentração celular (Xm) encontrada foi de 1588 ± 11 mg.L-1 com uso de 20 mM de NaNO3. O uso do processo descontínuo alimentado, o qual teve adição de 20 mM de NaNO3 feito num intervalo a cada 48 horas sendo iniciada a adição no primeiro dia, permitiu a obtenção de Xm = 2753 ± 7 mg.L-1; porém não foi possível eliminar a fase lag do cultivo, levando a uma produtividade em células (Px) de 351 ± 1 mg.L-1.dia-1. O processo semi-contínuo foi eficiente para eliminar a fase lag do cultivo, permitindo a obtenção de Xm = 2399 ± 5 mg.L-1 e um aumento de até 50% em Px, que chegou a valores de 525 ± 1 mg.L-1.dia-1 em cultivos com uso de 20 mM de NaNO3. Nesta condição os teores de proteínas e lipídios nas biomassas foram de 34,8 ± 0,2% e 38,6 ± 0,2%, respectivamente. Foi observado que, independentemente do tipo de processo empregado, há um decréscimo do valor do fator de conversão de nitrogênio em células (YX/N) com o aumento da adição de NaNO3. O maior valor de YX/N foi obtido no experimento com processo semi-contínuo e uso de 2 mM de NaNO3 no meio de cultivo, com valor médio de 29,1 ± 0,1 mg mg-1 ao final do segundo ciclo. Porém, nesta condição, o teor de proteínas da biomassa foi de 17,3 ± 0,4%. Já os maiores valores de YX/N encontrados nos processos descontínuo e descontínuo alimentado foram, respectivamente, de 22,5 ± 1,6 e 7,1 ± 0,1 mg mg-1. Os resultados obtidos neste trabalho evidenciam o potencial de Ankistrodesmus braunii como fonte de proteínas e lipídios para uso industrial.

Kitāb Ṭabaqāt al-aqṭāb fī dhikr awliyāʼ Allāh taʻālá wa-aqwālihim wa-aḥwālihim ilá intihāʼ ājālihim / taʼlīf al-Shaykh al-Imām al-ʻĀlim al-ʻAllāmah Taqī al-Dīn al-Subkī : manuscript, 1567

A biographical dictionary of sufis.

Oxygen microprofiles and porewater chemistry in sediments of the Skagerrak

Sediment porewater oxygen profiles were measured with micro and needle electrodes in sediment cores of 27 stations in the Skagerrak (northeastern North Sea). Oxygen penetration depth ranged from 3 to 20 mm depth. Fluxes estimated from the oxygen gradients varied from 3 to 18 mmol m**-2 d**-1. Oxygen penetration and flux depend on water depth, but possibly more on the hydrological conditions, related to the import of fresh organic matter by primary production in the water column. Oxygen fluxes were not related to the total organic carbon (TOC) content of the sediments. Stations in the eastern part of the Skagerrak showed high burial rates of TOC. At 6 stations porewater chemistry of Fe, Mn and NO3- was strongly associated with the oxygen distribution. The average relative contribution of terminal electron acceptors to carbon mineralisation was estimated at 85% for O2, 0.5% for Mn, 4.5% for [NO3]3-, 1% for Fe and 9% for [SO4]2-. At one station the occurrence of exceptionally high solid manganese oxyhydroxides was probably related to an active internal manganese cycle.

(Table 1) Compositional variation with depth in a nodule of sediment sample RVG-10D

A detailed study of a nodule from the Somali Basin dated by 230Thexcess was correlated with the paleoceanographic events recorded in Site 236 (Leg 24) Deep Sea Drilling Project (DSDP) cores. Tentative indications are that the phase of nodule accretion starting with the development of pillar structure at a depth of 20 mm in the nodule around 13 Ma coincides with increased Antarctic Bottom Water (AABW) flow and an elevated calciumcarbonate compensation depth (CCD). The Late Miocene lowering of the CCD is represented by the mottled zones between 8 and 18 mm in the nodule is characterised by an abundant silicate component (>20%) of aeolian origin. The Miocene/Pliocene boundary (5 Ma) occurs at a depth of about 8 mm and is represented by the development of pillar structure and a minimum of aeolian dust (10.3%). The increased biological productivity of the Somali surface water since the Middle Miocene is demonstrated by the increasing Corg content of the nodule (from 0.11 to 0.19%) towards its surface.

(Table 1, page 168) Iron, manganese, and phosphorus concentrations in Fe-Mn-concretions from the Gulf of Bothnia, northern Baltic Sea

Fe-Mn-concretions of a spheroidal type were found according to electron probe determinations to consist of alternating iron- and manganese-rich layers. This pattern was ascribed to seasonal variations in the physico-chemical conditions governing the precipitation of the hydrous oxides of iron and manganese. Calculations based on the rhythmic growth of the concretions investigated gave a mean accumulation rate of 0.15-0.20 mm/yr. The rather high phosphorus content (average 3.5 % P2O5) of the concretions was found to be concentrated in the iron-rich layers, probably as a result of the scavenging effect of ferric hydroxide.

Distribution of planktic foraminifera in surface sediments and sedimentation rates of sediment cores from the Ibero-Moroccan continental rise and slope

Cores from the Atlantic Ibero-Moroccan continental rise and slope contain fine-grained Late Pleistocene and Holocene sediments. These young sediments cover the continental margin in large lensformed litho- and biostratigraphically well-defined units. The total sedimentation rates range from 4 cm/ 1000 yrs. to 27 cm/1000 yrs. off Portugal and from 6 cm/1000 yrs. to 14 cm/1000 yrs. off Morocco. Only a small proportion of these sediments usually consists of sand-sized particles (>0.063 mm) which are mostly dominated by foraminifera. Both planktonic and benthic foraminifera are much more abundant in Late Pleistocene and Holocene samples from the upper slope in comparison to those from the deeper slope and from the abyssal plains. Total sedimentation rates during cool and warm climatic stages are rather similar for both groups of foraminifera. For example, in Late Holocene sediments 7 x 10**3 benthic and 201 x 10**3 planktonic foraminifera (fraction 0.63 -0.20 mm) per 100 cm**2 and 1000 yrs. are preserved in the Tagus, 10-19 X 10**3 benthic and about 1.3 X 10**6 planktonic foraminifera are preserved in the Seine abyssal plain sediments. Values from the upper slope sediments are higher for benthic foraminifera by a factor of 60 off Portugal and 60 to 70 off Morocco. The values for planktonic ones differ by factors of 6-12 and 6 respectively. These numbers seem to reflect differences in production and preservation. Production rates of planktonic foraminifera generally seem to be somewhat higher during Holocene than during Late Pleistocene, and the rates of benthic foraminifera appear rather higher during Late Pleistocene than during Holocene.

Seafloor magnetic anomalies and ages observed from shipboard and airborne measurements from New Zealand to the Amundsen Sea (south of 64°S)

Geophysical data acquired using R/V Polarstern constrain the structure and age of the rifted oceanic margin of West Antarctica. West of the Antipodes Fracture Zone, the 145 km wide continent-ocean transition zone (COTZ) of the Marie Byrd Land sector resembles a typical magma-poor margin. New gravity and seismic reflection data indicates initial continental crust of thickness 24 km, that was stretched 90 km. Farther east, the Bellingshausen sector is broad and complex with abundant evidence for volcanism, the COTZ is ~670 km wide, and the nature of crust within the COTZ is uncertain. Margin extension is estimated to be 106-304 km in this sector. Seafloor magnetic anomalies adjacent to Marie Byrd Land near the Pahemo Fracture Zone indicate full-spreading rate during c33-c31 (80-68 Myr) of 60 mm/yr, increasing to 74 mm/yr at c27 (62 Myr), and then dropping to 22 mm/yr by c22 (50 Myr). Spreading rates were lower to the west. Extrapolation towards the continental margin indicates initial oceanic crust formation at around c34y (84 Myr). Subsequent motion of the Bellingshausen plate relative to Antarctica (84-62 Myr) took place east of the Antipodes Fracture Zone at rates <40 mm/yr, typically 5-20 mm/yr. The high extension rate of 30-60 mm/yr during initial margin formation is consistent with steep and symmetrical margin morphology, but subsequent motion of the Bellingshausen plate was slow and complex, and modified rift morphology through migrating deformation and volcanic centers to create a broad and complex COTZ.

Ablative Pyrolysis of Beetle-killed Trees

Thesis (Ph.D.)--University of Washington, 2016-06

High glucose-mediated effects on endothelial cell proliferation occur via p38 MAP kinase

The mitogen-activated protein ( MAP) kinases contribute to altered cell growth and function in a variety of disease states. However, their role in the endothelial complications of diabetes mellitus remains unclear. Human endothelial cells were exposed for 72 h to 5 mM ( control) or 25 mM ( high) glucose or 5 mM glucose plus 20 mM mannitol ( osmotic control). The roles of p38 and p42/44 MAP kinases in the high glucose-induced growth effects were determined by assessment of phosphorylated MAP kinases and their downstream activators by Western blot and by pharmacological inhibition of these MAP kinases. Results were expressed as a percentage ( means +/- SE) of control. High glucose increased the activity of total and phosphorylated p38 MAP kinase ( P < 0.001) and p42/44 MAP kinase ( P < 0.001). Coexposure of p38 MAP kinase blocker with high glucose reversed the antiproliferative but not the hypertrophic effects associated with high-glucose conditions. Transforming growth factor (TGF)-beta1 increased the levels of phosphorylated p38 MAP kinase, and p38 MAP kinase blockade reversed the antiproliferative effects of this cytokine. The high glucose-induced increase in phosphorylated p38 MAP kinase was reversed in the presence of TGF-beta1 neutralizing antibody. Although hyperosmolarity also induced antiproliferation (P < 0.0001) and cell hypertrophy (P < 0.05), there was no change in p38 activity, and therefore inhibition of p38 MAP kinase had no influence on these growth responses. Blockade of p42/44 MAP kinase had no effect on the changes in endothelial cell growth induced by either high glucose or hyperosmolarity. High glucose increased p42/44 and p38 MAP kinase activity in human endothelial cells, but only p38 MAP kinase mediated the antiproliferative growth response through the effects of autocrine TGF-beta1. High glucose-induced endothelial cell hypertrophy was independent of activation of the MAP kinases studied. In addition, these effects were independent of any increase in osmolarity associated with high-glucose exposure.

Adventitious root formation in cuttings of Backhousia citriodora F. Muell 2. Seasonal influences of temperature, rainfall, flowering and auxins on the stock plant

A 2-year study was carried out on established trees at two sites in southeastern Queensland, Australia, to identify environmental factors that influenced rooting of Backhousia citriodora from cuttings. Complex interactions of rainfall events above 20 mm from the preceding month and mean maximum temperature on stock plants resulted in a correlation with rooting success of r = 0.81 and 0.74 for sites at The University Of Queensland, Gatton Campus, and Cedar Glen, respectively. A more detailed investigation under controlled environmental conditions showed that maintaining stock plants at temperatures between 10 and 30degreesC had no direct effect on rooting capacity. However, temperature was correlated with growth, which may have an indirect effect on root formation. In spring floral initiation was found to only delay rooting and had no effect on the final rooting percentage. A series of seasonal experiments demonstrated that application of the auxins indole-3-acetic acid, indole-3-butyric acid and napthaleneacetic acid over a range of concentrations (1000-8000 mug/ml) did not significantly increase rooting compared to the control and there is no practical advantage in applying auxins. Seasonal results and the temperature experiment also suggest that under a glasshouse environment with higher temperatures in winter and an adequate supply of water, B. citriodora cuttings can be successfully rooted over the whole year. (C) 2004 Elsevier B.V. All rights reserved.