Defining natural factors that stimulate and inhibit cellulose:xyloglucan hetero-transglucosylation

Certain transglucanases can covalently graft cellulose and mixed-linkage β-glucan (MLG) as donor substrates onto xyloglucan as acceptor substrate and thus exhibit cellulose:xyloglucan endotransglucosylase (CXE) and MLG:xyloglucan endotransglucosylase (MXE) activities in vivo and in vitro. However, missing information on factors that stimulate or inhibit these hetero-transglucosylation reactions limits our insight into their biological functions. To explore factors that influence hetero-transglucosylation, we studied Equisetum fluviatile hetero-trans-β-glucanase (EfHTG), which exhibits both CXE and MXE activity, exceeding its xyloglucan:xyloglucan homo-transglucosylation (XET) activity. Enzyme assays employed radiolabelled and fluorescently labelled oligomeric acceptor substrates, and were conducted in vitro and in cell walls (in situ). With whole denatured Equisetum cell walls as donor substrate, exogenous EfHTG (extracted from Equisetum or produced in Pichia) exhibited all three activities (CXE, MXE, XET) in competition with each other. Acting on pure cellulose as donor substrate, the CXE action of Pichia-produced EfHTG was up to approximately 300% increased by addition of methanol-boiled Equisetum extracts; there was no similar effect when the same enzyme acted on soluble donors (MLG or xyloglucan). The methanol-stable factor is proposed to be expansin-like, a suggestion supported by observations of pH dependence. Screening numerous low-molecular-weight compounds for hetero-transglucanase inhibition showed that cellobiose was highly effective, inhibiting the abundant endogenous CXE and MXE (but not XET) action in Equisetum internodes. Furthermore, cellobiose retarded Equisetum stem elongation, potentially owing to its effect on hetero-transglucosylation reactions. This work provides insight and tools to further study the role of cellulose hetero-transglucosylation in planta by identifying factors that govern this reaction.     

Characterization of the cytochrome c oxidase in isolated and purified plasma membranes from the cyanobacterium Anacystis nidulans

Functionally intact plasma membranes were isolated from the cyanobacterium (blue-green alga) Anacystis nidulans through French pressure cell extrusion of lysozyme/EDTA-treated cells, separated from thylakoid membranes by discontinuous sucrose density gradient centrifugation, and purified by repeated recentrifugation. Origin and identity of the chlorophyll-free plasma membrane fraction were confirmed by labeling of intact cells with impermeant protein markers, [35S]diazobenzenesulfonate and fluorescamine, prior to membrane isolation. Rates of oxidation of reduced horse heart cytochrome c by purified plasma and thylakoid membranes were 90 and 2 nmol min-1 (mg of protein)-1, respectively. The cytochrome oxidase in isolated plasma membranes was identified as a copper-containing aa3-type enzyme from the properties of its redox-active and EDTA-resistant Cu2+ ESR signal, the characteristic inhibition profile, reduced minus oxidized difference spectra, carbon monoxide difference spectra, photoaction and photodissociation spectra of the CO-inhibited enzyme, and immunological cross-reaction of two subunits of the enzyme with antibodies against subunits I and II, and the holoenzyme, of Paracoccus denitrificans aa3-type cytochrome oxidase. The data presented are the first comprehensive evidence for the occurrence of aa3-type cytochrome oxidase in the plasma membrane of a cyanobacterium similar to the corresponding mitochondrial enzyme (EC 1.9.3.1).     

Application of Urine Analysis to Diagnosis and Treatment of Heroin Addiction

Experience with urine analysis for morphine using thin-layer chromatography in 310 cases of real or possible heroin abuse showed that it was valuable not only in detecting improper drug use but also in monitoring treatment. The results of this test can be available routinely in 24, and exceptionally in five hours. A negative result implies that the subject has taken less than 10 mg. of heroin in the past 24 hours.     

Cell adhesion and acquisition of detergent resistance by the cytoskeleton of cultured chick fibroblasts

About 30% of the proteins of adherent cultured chick embryo fibroblasts are not solubilized by the non-ionic detergent Triton X-100 and remain firmly attached to the substratum. The insoluble residue contains a considerable part of the cell's cytoskeleton and its major constituents are large external transformation-sensitive (LETS) protein, the heavy chain of myosin, a 52,000 molecular weight protein and actin. Kinetic studies reveal that cytoskeleton insolubility in Triton is acquired either concurrently with cell adhesion or very closely with it. Neither cell adhesion nor binding of the Triton cytoskeleton to the substratum require de novo synthesis of protein. In the attempt to assess the role of LETS protein in cytoskeleton attachment, we find that trypsin-detached cells rapidly acquire Triton-insoluble cytoskeleton although their LETS protein content is about 15--20% of its level in long-term cultures. Removal of the great majority of LETS molecules of adherent cultures by either urea or trypsin treatment does not affect the relative amount or composition of the anchored cytoskeletal proteins. Also, LETS protein of cultures exposed to cycloheximide for extended periods of time, is reduced to 10% of its maximum amount without much affecting the attachment and composition of the cytoskeleton. It is deduced that the great majority of LETS protein is not required for the attachment of the Triton cytoskeleton to the substratum.     

Alterations of the carrier-mediated transport of an anionic solute,methotrexate, by charged liposomes in Ehrlich ascites tumor cells

Summary Interaction of positively charged liposomes with Ehrlich ascites tumor cells increases the bidirectional transmembrane fluxes of the anionic folic acid analog, methotrexate. Negative liposomes reduce methotrexate influx. Stimulation of methotrexate influx by positively charged liposomes is time and concentration dependent, requiring at least a 5-min incubation with 2.5mm phosphatidylcholine containing 20% stearylamine for maximum effect. Stimulation is not appreciably reversed by washing the cells. Similar increases are observed for influx and efflux so that there is no change in the steady-state methotrexate electrochemical-potential difference across the cell membrane. The increase in influx appears to be a stimulation of the carrier-mediated transport process for methotrexate since both control and stimulated influx are abolished by the competitive inhibitor, 5-formyltetrahydrofolate or the sulfhydryl group inhibitor,p-chloromercuriphenylsulfonic acid and the Q₁₀ of the system remains unchanged. Influx of 5-methyltetrahydrofolate, which shares the same transport carrier as methotrexate, is also stimulated. However, the transport of folic acid, which is structurally similar to methotrexate but does not utilize the carrier, is unaffected. The kinetic change induced by positively charged liposomes is an increase in theV _ma ⁱⁿ , while theK _t ⁱⁿ remains unchanged. Trans-stimulation of methotrexate influx by 5-formyltetrahydrofolate occurs to the same extent in the presence or absence of positively charged liposomes. The liposomes have no apparent effect on the intracellular water, the extracellular space, or the chloride distribution ratio. The data suggest that interaction of positively charged liposomes with Ehrlich ascites tumor cells accelerates the rate of transposition of the membrane carrier system for methotrexate, altering the kinetics of transport without a change in transport thermodynamics.     

Trends in tuna carbon isotopes suggest global changes in pelagic phytoplankton communities

Considerable uncertainty remains over how increasing atmospheric CO₂ and anthropogenic climate changes are affecting open‐ocean marine ecosystems from phytoplankton to top predators. Biological time series data are thus urgently needed for the world's oceans. Here, we use the carbon stable isotope composition of tuna to provide a first insight into the existence of global trends in complex ecosystem dynamics and changes in the oceanic carbon cycle. From 2000 to 2015, considerable declines in δ¹³C values of 0.8‰–2.5‰ were observed across three tuna species sampled globally, with more substantial changes in the Pacific Ocean compared to the Atlantic and Indian Oceans. Tuna recorded not only the Suess effect, that is, fossil fuel‐derived and isotopically light carbon being incorporated into marine ecosystems, but also recorded profound changes at the base of marine food webs. We suggest a global shift in phytoplankton community structure, for example, a reduction in ¹³C‐rich phytoplankton such as diatoms, and/or a change in phytoplankton physiology during this period, although this does not rule out other concomitant changes at higher levels in the food webs. Our study establishes tuna δ¹³C values as a candidate essential ocean variable to assess complex ecosystem responses to climate change at regional to global scales and over decadal timescales. Finally, this time series will be invaluable in calibrating and validating global earth system models to project changes in marine biota.     

A reliable method for assessing rotational power

Rotational core training is said to be beneficial for rotational power athletes. Currently, there has been no method proposed for the reliable assessment of rotational power. Therefore, our purpose was to determine the test-retest reliability of kinetic and kinematic rotational characteristics of a pulley system when performing a rotational exercise of the axial skeleton in the transverse plane to find out if this would be a reliable tool for evaluating rotational power. Healthy, college-aged men (n = 8) and women (n = 15) reported for 3 testing sessions. The participants were seated on a box, and they held the handle with both arms extended in front of their body, starting their motion with their torso rotated toward the machine. All the participants rotated their torso forcefully until they reached 180° of rotation, and they then slowly returned to the starting position, 3 times per trial, with 3 loads: 9% body weight (BW), 12% BW, and 15% BW. The repetition with the greatest power for each trial for each load was analyzed. The mean peak power repetition (watts) for all the subjects was 20.09 ± 7.16 (9% BW), 26.17 ± 8.6 (12% BW), and 30.74 ± 11.022 (15% BW) in the first training session and 22.3 ± 8.087 (9% BW), 28.7 ± 11.295 (12% BW), and 33.52 ± 12.965 (15% BW) in the second training session with intraclass correlation coefficients of 0.97 (9%BW), 0.94 (12%BW), and 0.95 (15%BW). When the participants were separated by sex, there were no significant differences between groups. Based on these results, it was found that a pulley system and an external dynamometer can be used together as a reliable research tool to assess rotational power.