[125I]calmodulin binding to synaptic plasma membrane from rat brain: Kinetic and arrhenius analysis

Binding of [¹²⁵I]calmodulin was characterized in highly purified synaptic plasma membrane (SPM) prepared from rat brain. By Scatchard analysis, the Ca²⁺-dependent membrane binding of [¹²⁵I]calmodulin was found to have a B_max of 284 pmol/mg protein and an apparent affinity with a K_d of 131 nM. Kinetic analysis indicates that at 37°C, the dissociation of [¹²⁵I]calmodulinmembrane complexes follows first-order reaction and consists of two components: a dissociation constant (k) of 3.7×10^–1 min^–1 and a half-time (t_1/2) of 1.8 min for the fast component, and a k of 4.8×10^–2 min^–1 and a t_1/2 of 14.5 min for the slow component. At 0°C, substantial dissociation still occurred, with a k of 4.5×10^–2 min^–1 and a t_1/2 of 15.3 min for the fast component, and a k of 5.5×10^–3 min^–1 and a t_1/2 of 125.5 min for the slow component. These data on binding affinity and dissociation kinetics are consistent with the notion that SPM can readily and rapidly associated and dissociate calmodulin. In Arrhenius analysis of temperature effects, [¹²⁵I]calmodulin binding to SPM exhibits a biphasic function, with the transition temperature (T_d) estimated to be 23.8°C, suggesting that binding is influenced by lipid phase transition of the membrane. The binding of [¹²⁵I]calmodulin to the synaptic membrane was found to be increased by corticosterone (10^–7–10^–6 M), a steroid hormone, and decreased by ethanol (50–200 mM), a centrally acting drug. Our data on the characteristics of calmodulin binding to the SPM provide groundwork for future studies on physiological and pharmacological regulation of calmodulin translocation to and from the plasma membrane in synaptic terminals.Abbreviations used CaM calmodulin - SPM synaptic plasma membrane - ATPase adenosine triphosphatase - Tris tris(hydroxymethyl)aminomethane - EGTA ethylene-bis(oxyethylenenitrilo)tetraacetic acid - SDS sodium dodecyl sulfate - TFP trifluoperazine - K_d dissociation constant - B_max maximum binding - k first-order rate constant - t_1/2 half-time - T_d transition temperature     

Characteristics and Regulatory Properties of the H+-ATPase in a Plasma Membrane Fraction Purified from Arabidopsis thaliana

The aqueous two-phase partitioning technique was utilized to isolate a plasma membrane (PM) fraction from etiolated seedlings of Arabidopsis thaliana. The purification procedure adopted yielded a fraction highly enriched in PM as compared to inner membranes, with a recovery of about 30%, as judged from the activities of PM markers such as vanadate-sensitive ATPase, FC binding and UDP-glucose sterol glucosyltransferase. The purified PM fraction displayed vanadate-sensitive H⁺ pumping activity. Its purity was confirmed by the biochemical characteristics of its ATPase activity assayed in the absence of Ca²⁺: sensitivity to vanadate (IC₅₀ ca. 1 μM), Mg²⁺-dependence, insensitivity to molybdate, oligomycin and nitrate, pH optimum at 6.6. The PM H⁺-ATPase activity was stimulated by fusicoccin and by a controlled treatment of the PM with trypsin. In both cases stimulation was much stronger on the activity assayed at pH 7.5 than on the activity at pH 6.6. Moreover, neither fusicoccin nor the treatment with trypsin stimulated the portion of activity (30 to 40% at pH 7.5) which decayed upon preincubation of the PM in assay medium without ATP.     

Measurement of trace elements in murine liver tissue samples: Comparison between ICP-MS/MS and TXRF

BackgroundTrace elements exhibit essential functions in many physiological processes. Thus, for research focusing on trace element homeostasis and metabolism analytical methods allowing for multi-element analyses are fundamental. Small sample amounts may be a big challenge in trace element analyses especially if also other end points want to be addressed in the same sample. Therefore, the aim of the present study was to examine trace elements (iron, copper, zinc, and selenium) in murine liver tissue prepared by a RIPA buffer-based lyses method.Methods and resultsAfter centrifugation, lysates and pellets were obtained and trace elements were analyzed with TXRF in liver lysates. The results were compared to that obtained by a standard microwave-assisted acidic digestion with subsequent ICP-MS/MS analysis of the same liver tissue, liver lysates, and remaining pellets. In addition, trace element concentrations, determined in murine serum with both methods, were compared. For serum samples, both TXRF and ICP-MS/MS provide similar and highly correlating results. Furthermore, in liver lysate samples prepared with RIPA buffer, comparable trace element concentrations were measured by TXRF as with the standard digestion technique and ICP-MS/MS. Only marginal amounts of trace elements were detected in the pellets.ConclusionTaken together, the results obtained by the present study indicate that the RIPA buffer-based method is suitable for sample preparation for trace element analyses via TXRF, at least for the here investigated murine liver samples.     

D-amino acid levels in human physiological fluids

Plasma, urine, cerebrospinal fluid (CSF), and amniotic fluid were examined to determine whether free D-amino acids were present and if so at what levels. It was found that D-amino acids exist in all physiological fluids tested, but that their level varied, considerably. The lowest levels of D-amino acids were usually found in amniotic fluid or CSF (almost always <1% of the corresponding L-amino acid). The highest levels were found in urine (usually tenth percent to low percent levels). Pipecolic acid seemed to be different from the other amino acids tested in that it was excreted primarily as the D-enantiomer (often >90%). Correspondingly high levels of D-pipecolic acid were not found in plasma. Some of the trends found in this work seemed to be analogous to those found in a recent rodent study. © 1993 Wiley-Liss, Inc.     

A Meaningful Attempt: Applying Dielectric Barrier Discharge Plasma to Induce Polarization of Macrophages

Macrophage polarization plays an important role in many macrophage-related diseases. This study was designed to preliminarily explore the effects of dielectric barrier discharge (DBD) plasma on the polarization direction and cell activity of macrophages with different phenotypes (ie, M0, M1, and M2). The M1 macrophage marker inducible nitric oxide synthase (iNOS) and M2 macrophage marker cluster of differentiation 206 (CD206) were detected by western blot (WB). The effects of DBD plasma on macrophage viability were analyzed by using a cell counting kit-8 detection kit. M0, M1, and M2 macrophages exhibited a decrease in iNOS expression and an increase in CD206 expression after the DBD plasma intervention. Additionally, the decrease in macrophage viability remained non-significant after initiating the intervention. DBD plasma can promote the transformation of M0 and M1 macrophages to M2 macrophages, and can further enhance the expression of the M2 macrophage phenotype marker CD206. Our study not only demonstrates the potential therapeutic value of DBD plasma for macrophage-related diseases, but it also provides a new direction for research to improve the treatment of macrophage-related diseases. © 2023 Bioelectromagnetics Society.     

油藏注入水靶向脱硫潜力的高温脱硫菌群富集驯化

【背景】海上油田见聚后产出水硫化物超标,影响到注聚水的配聚黏度,采用生物脱硫时,由于常规除硫菌难以适应除油后产出液的高温,使得脱硫效果不佳。【目的】分析海上采出液水处理过程的菌群结构,明确生物处理各节点的菌群构成变化;开展耐高温脱硫菌驯化筛选,获得耐高温的高效脱硫菌。【方法】采集来自胜利油田海三站的水样,以16S rRNA基因高通量测序技术分析样本菌群结构,并分别在不同温度(55、60和65℃)下的无机富集培养基中进行多轮转接驯化,结合常压室温等离子体(atmospheric and room temperature plasma, ARTP)诱变技术筛选获得耐高温的脱硫菌群,采用宏基因组测序技术分析富集菌群的组成,并测定其脱硫能力。【结果】处理前的采出液水样含有较多的嗜热菌和硫酸盐还原菌,如Thermodesulfovibrio、Pseudothermotoga、Thermolithobacter、Fervidobacterium、Thermovenabulales和Pseudomonas;以厌氧气浮除油工艺处理的出水中,嗜氢菌属(Hydrogenophilus)成为最主要的优势菌,...     

Expression of p21ras-related protein in the plasma and tissue of patients with adenomas and carcinomas of the colon

Over-expression of p21 ras-related protein was determined in the plasma by immunoblotting and in the tissue by immuno-histochemistry in a cohort of patients undergoing colonoscopy. In the plasma samples, p21 ras over-expression was detected in: 9% (4/47) of normal controls; 21% (13/61) of individuals with normal colonoscopies but with a prior history of colonic neoplasia; 12% (4/33) of small adenoma patients, 29% (6/21) of large adenoma patients; 63% (5/8) of carcinoma-in-adenoma patients; 50% (2/4) of Dukes' A carcinoma patients; and 20% (2/10) of Dukes' B-D carcinoma patients. In the tissue samples, p21 ras over-expression was detected in: 25% (2/8) of small adenoma patients; 44% (4/9) of large adenoma patients; 100% (4/4) of carcinoma-in-adenoma patients; and 33% (1/3) of Dukes' B-C carcinoma patients. For matched plasma-tissue pairs, there was a statistically significant correlation for p21 ras over-expression (R = 0.47, p = 0.02).     

Distribution of Plasma Membrane H+-ATPase and Polar Current Patterns in Leaves and Stems of Elodea canadensis*

The proton pumping ATPase in the plasma membrane of Elodea canadensis is believed to play a major role in inorganic carbon acquisition. To investigate potentially different carbon uptake strategies within the same plant, plasma membrane H⁺-ATPase distribution and polar current patterns were investigated in Elodea leaves and stems. Specific activity of plasma membrane H⁺-ATPase in leaf microsomal fractions was tenfold higher than in stem derived microsomes. Probing western blots with a monoclonal antibody specific for plasma membrane H⁺-ATPase, yielded strongly visible double bands at 100 kDa in leaf microsome preparations, whereas little antigen was detected in analogous stem microsome preparations. Using the same plasma membrane H⁺-ATPase specific antibody on tissue sections, the enzyme was found almost exclusively localized at the border of cells at the lower leaf surface. A positive ion current leaving the lower leaf surface was measured, using a vibrating probe device. Part of this current entered the upper leaf surface and part of it the internodes of the stem. The experimental results support the view, that Elodea leaves have different means of inorganic carbon uptake than stem internodes.     

Identification and partial characterization of α2-macroglobulin from the tuatara (Sphenodon punctatus)

α₂-Macroglobulin (α₂-M), a large molecular mass proteinase-binding protein, was identified in plasma from tuatara (Sphenodon), a rare reptile endemic to New Zealand. In this genus, α₂-M constitutes 11–13% of total plasma protein (∼2.2–3.9 mg/ml). Analysis of blood samples collected at approximately monthly intervals from individual tuatara indicated that the plasma level of α₂-M remains fairly constant. The subunits of tuatara α₂-M have an apparent molecular mass of ∼160 kDa as determined by SDS-polyacrylamide gel electrophoresis and the intact protein is an oligomer that contains inter-chain disulfide bonds. N-terminal sequence analyses of tuatara α₂-M revealed a distinct similarity to α-macroglobulins of other vertebrates and that at least two types of α₂-M subunits are present in plasma of tuatara.     

Ehrlich cell plasma membrane redox system is modulated through signal transduction pathways involvingcGMP and Ca2+ as second messengers

Ehrlich cell plasma membrane ferricyanide reductase activity increased in the presence of mastoparan, a generic activator of G proteins, using either whole cells or isolated plasma membrane fractions. Agents that increase intracellularcAMP also increased the rate of ferricyanide reduction by Ehrlich cells. For the first time, evidence is shown on a modulation of plasma membrane redox system bycGMP. In fact, permeant analogs ofcGMP, dibutyrylcGMP, and 8-bromo-cGMP increased the rate of ferricyanide reduction by the Ehrlich cell plasma membrane redox system. Furthermore, specific inhibition ofcGMP-phosphodiesterases by dipyridamole was also accompanied by an enhancement in the rate of ferricyanide reduction. On the other hand, treatments expected to increase cytoplasmic Ca²⁺ concentrations were accompanied by a remarkable stimulation of the reductase activity. Taking all these data together, it seems that the Ehrlich cell plasma membrane redox system is under a multiple and complex regulation by different signal transduction pathways involving G proteins, cyclic nucleotides, and Ca²⁺ ions.