Correction for light absorption in fluorescence studies of protein-ligand interactions

It is shown that absorption of the excitation light can lead to substantial systematic errors in fluorescence measurements of equilibrium constants for formation of protein-ligand complexes. The assumptions about the optical arrangement of the fluorescence spectrometer involved in the calculation of the correction of this absorption are discussed. A general semiempirical correction procedure which can be used for (calculated) absorbance values as high as 5 is described. The importance of choosing the excitation wavelength so as to minimize the necessity for these corrections is emphasized.     

离体外翻肠囊法研究白及提取物中6种成分的肠吸收特性

为研究白及提取物中4-(葡萄糖氧基)-肉桂酸葡萄糖氧基苄酯(B12)、2-异丁基苹果酸(B6)、1-[4-(葡萄糖氧)苄基]-2-异丁基苹果酸酯(B17)、1,4-二[4-(葡萄糖氧)苄基]-2-异丁基苹果酸酯(B14)、二氢菲1(B19)和1,4-二[4-(葡萄糖氧)苄基]-2-异丁基苹果酸酯-2-[4-O-肉桂酰基-6-O-乙酰基]葡萄糖苷(B23)6个成分在大鼠小肠中的吸收动力学特性。实验采用大鼠离体外翻肠囊模型考察各成分在不同浓度、不同肠段的吸收特性,建立超高效液相色谱-三重四极杆串联质谱联用仪(UPLC-MS/MS)法测定各成分含量,计算累计吸收量和吸收速率常数。结果表明,除B6成分的低浓度外,各成分在不同浓度、不同肠段下均表现为线性吸收,其回归相关系数(R)均达到0.9以上,符合一级吸收速率,且吸收速率常数随着浓度的增加而增加,提示各成分吸收机制为被动吸收;在同一浓度下不同肠段的总体吸收趋势为十二指肠的吸收要大于回肠、结肠和空肠。综上,白及提取物中6种成分在小肠中均有吸收,但小肠对各成分的吸收具有选择性。本研究可为白及提取物的药物临床开发,确定药物剂型方面提供了一定的实验参考。     

The role of the heme distal ligand in the post-translational modification of Synechocystis hemoglobin

Synechocystis sp. PCC 6803 hemoglobin is a cyanobacterial Group I truncated hemoglobin. In the absence of an exogenous ligand, its single heme group is coordinated by His46 (E10, distal) and His70 (F8, proximal). The protein can undergo a post-translational modification by which His117 (H16, in the C-terminal helix) reacts with the heme 2-vinyl group to form a Markownikoff adduct. The new C-N bond prevents heme loss, alters the dynamics of the protein, and influences ligand binding to the heme group. To explore the factors conditioning the formation of the cross-link, variants of the protein that contained an alanine or a leucine at position 46 (E10) were prepared. A double replacement (His46Leu and Tyr22 (B10) to Phe) was also performed to perturb the network of interactions stabilizing bound exogenous ligand. The single and double replacements affected the optical and NMR properties of the globin, each in a different fashion. Heme-protein cross-linking, as promoted by sodium dithionite, was retarded by the replacement of His46, but reactivity was recovered when imidazole or cyanide was used as exogenous ligand. In addition, a significant amount of a second product was systematically obtained when dithionite treatment was performed on the cyanide-bound proteins. This species was identified by NMR spectroscopy to be an adduct to the 4-vinyl group. It was concluded that the specificity and rate of the cross-linking reaction depended critically on the nature of the sixth ligand to the heme iron.     

Inhibition of copper absorption by zinc

Copper and zinc interact at the intestinal mucosal level, affecting copper absorption. Amino acids, such as histidine, may affect the absorption of these two elements by chelating these cations. The two mechanisms could have additive potential. This possibility was investigated using a duodenal-jejunal single-pass perfusion procedure in anesthetized rats. Copper absorption and tissue retention from solutions containing 0.1 mM copper were determined in the presence of either no zinc or equimolar zinc, or at a zinc/copper ratio of 10/1, either without histidine or with histidine at a 10/1 or 20/1 ratio to copper. Copper removal from the intestinal lumen was decreased by zinc, and further reduced by increasing concentrations of histidine. There was a greater accumulation of copper in the small intestine, reaching a maximum with a 10-fold excess of histidine. With zinc at a 10/1 ratio to copper, the addition of a 10- or 20-fold molar excess of histidine further decreased the net uptake of copper from the perfusate while greater copper accumulation in the tissue occurred. Histidine thus enhances the inhibitory effects of zinc on copper absorp|tion, suggesting the application of convergent mechanisms for diminishing copper uptake. This could be relevant for the treatment of Wilson’s disease.     

Temperature dependence in the absorption spectra of beef liver catalase

The spin characteristics of the ferric heme groups in native beef liver catalase. and in the complexes formed by reaction with fluoride, cyanide, azide, thiocyanate, and cyanate ions have been studied using absorption spectroscopy over the temperature range of 4–285 K. The azide. isothiocyanate, and isocyanate complexes of catalase are considered to be high-spin ferric heme complexes at room temperature, but undergo a thermal spin change below 300 K. The temperature dependence of these absorption spectra, however, cannot be analyzed in terms of simple Boltzmann distributions between two S = $\text{1}\text{2}$ and S = $\text{5}\text{2}$ spin stales. The data show that these spin changes occur over a very narrow temperature range, but do not result in the formation of completely, low-spin complexes. The data also suggest that the thermal spin changes that occur below the glassing temperature of the solvent are dependent upon the conformational changes which take place within the protein itself with a change in temperature, and which directly affect the environment of the heme group.     

Stability and absorption of chromium and absorption of chromium histidinate complexes by humans

Increased intake of chromium (Cr) often leads to improvements in glucose, insulin, lipids, and related variables in studies involving humans and experimental and farm animals. However, the results are often variable, depending not only on the selection of subjects but also dietary conditions and the form of supplemental Cr used. Our objective was to find a Cr supplement suitable for humans that was absorbed better than any of those available. Chromium absorption by six adult subjects, three males and three females, was determined based on the amount of Cr excreted in the urine in the initial 2 d following intake of 200 μg of Cr of the various forms of chromium tested. The absorption of the newly synthesized complexes was greatest for those containing histidine. Urinary Cr losses for six control subjects consuming 200 μg of Cr as Cr histidinate increased from basal levels of 256±48 to 3670±338 ng/d compared with 2082±201 ng for Cr picolinate, the currently most popular nutrient supplement, in the 48h following Cr consumption. Chromium histidinate complexes were stable and absorption was similar to the initial values after more than 2 yr. Mixing of some of the complexes with starch, which was postulated to improve Cr absorption, was shown to essentially block Cr absorption within 1 mo. These data demonstrate that urinary Cr losses need to be determined because stability and absorption of the Cr complexes varies widely and could be responsible for the variability in some of the Cr supplementation studies. Chromium ***DIRECT SUPPORT *** A02Q2015 00003 histidinate complexes are absorbed better than any of the Cr complexes currently available and need to be evaluated as Cr nutritional supplements.     

Kinetic studies of small molecule interactions with protein kinases using biosensor technology

Protein kinases are among the most commonly targeted groups of molecules in drug discovery today. Despite this, there are few examples of using surface plasmon resonance (SPR) for kinase inhibitor interaction studies, probably reflecting the need for better developed assays for these proteins. In this article, we present a general methodology that uses biosensor technology to study small molecule binding to eight different serine/threonine and tyrosine kinases. Mild immobilization conditions and a carefully composed assay buffer were identified as key success factors. The methodology package consists of direct binding studies of compounds to immobilized kinases, kinase activity assays to confirm inhibitory effects, detailed kinetic analyses of inhibitor binding, and competition assays with ATP for identification of competitive inhibitors. The kinetic assays resolve affinity into the rates of inhibitor binding and dissociation. Therefore, more detailed information on the relation between inhibitor structure and function is obtained. This might be of key importance for the development of effective kinase inhibitors.     

超声弹性成像技术及其应用

超声弹性成像作为一种新的超声成像方法,它通过获取有关组织弹性信息进行成像,弥补了X射线、超声成像(US)、磁共振成像(MRI)、计算机断层扫描(CT)等传统医学成像模态不能直接提供组织弹性的不足,具有无创、简单、便宜、容易应用等优点,被广泛应用于临床,成为目前医学弹性成像的一个研究热点。本文介绍了弹性成像基本的原理,详细阐述了血管内超声弹性成像、超声振动声成像、超声剪切波弹性成像、瞬时弹性成像、心肌弹性成像等不同激励方式下的几种超声弹性成像的基本原理、成像方法、国内外目前的研究现状、成像优缺点等,叙述了超声弹性成像在乳腺检测、前列腺癌的检测、射频消融监测和检测、冠状动脉粥样硬化的治疗等临床应用,并总结了超声弹性成像的发展趋势。     

Further studies on absorption changes arising in dye-stained nerves during excitation

Summary Changes in light absorption during nerve excitation (absorption responses) were detected from the crab leg nerve, the rabbit vagus, and the rat superior cervical ganglion (SCG) stained with a merocyanine-rhodanine. Dependences of the responses on the wavelength and polarization of the incident light (absorption spectra) showed characteristic features with the respective nerves. In the crab nerve, the pattern of response spectra was precisely analyzed based on the previously proposed scheme, which included the shift of absorption bands and the statistical reorientation of absorption oscillators of the dye molecules in the membrane matrix during nerve excitation. Different patterns of the response spectra between the crab nerve and the rabbit vagus suggested that distinct physicochemical environments of the dye occurred in these two classes of membranes. On the other hand, the characteristic pattern that arose in the rat SCG was explained by its morphological form, that is, unlike those in a bundle of axons, the membrane elements in the ganglion were randomly oriented with respect to the direction of the light polarization.     

Tetramer-dimer equilibrium of oxyhemoglobin mutants determined from auto-oxidation rates.

One of the main difficulties with blood substitutes based on hemoglobin (Hb) solutions is the auto-oxidation of the hemes, a problem aggravated by the dimerization of Hb tetramers. We have employed a method to study the oxyHb tetramer-dimer equilibrium based on the rate of auto-oxidation as a function of protein concentration. The 16-fold difference in dimer and tetramer auto-oxidation rates (in 20 mM phosphate buffer at pH 7.0, 37 degrees C) was exploited to determine the fraction dimer. The results show a transition of the auto-oxidation rate from low to high protein concentrations, allowing the determination of the tetramer-dimer dissociation coefficient K4,2 = [Dimer] 2/[Tetramer]. A 14-fold increase in K4,2 was observed for addition of 10 mM of the allosteric effector inositol hexaphosphate (IHP). Recombinant hemoglobins (rHb) were genetically engineered to obtain Hb with a lower oxygen affinity than native Hb (Hb A). The rHb alpha2beta2 [(C7) F41Y/(G4) N102Y] shows a fivefold increase in K4,2 at pH 7.0, 37 degrees C. An atmosphere of pure oxygen is necessary in this case to insure fully oxygenated Hb. When this condition is satisfied, this method provides an efficient technique to characterize both the tetramer-dimer equilibrium and the auto-oxidation rates of various oxyHb. For low oxygen affinity Hb equilibrated under air, the presence of deoxy subunits accelerates the auto-oxidation. Although a full analysis is complicated, the auto-oxidation studies for air equilibrated samples are more relevant to the development of a blood substitute based on Hb solutions. The double mutants, rHb alpha2beta2 [(C7) F41Y/(G4) N102A] and rHb alpha2beta2 [(C7) F41Y/(E10) K66T], show a lower oxygen affinity and a higher rate of oxidation than Hb A. Simulations of the auto-oxidation rate versus Hb concentration indicate that very high protein concentrations are required to observe the tetramer auto-oxidation rate. Because the dimers oxidize much more rapidly, even a small fraction dimer will influence the observed oxidation rate.     

Comparative studies on the interactions of dihydroartemisinin and artemisinin with bovine serum albumin using spectroscopic methods

The interactions of dihydroartemisinin (DHA) and artemisinin (ART) with bovine serum albumin (BSA) have been investigated using fluorescence, UV/vis absorption and Fourier transform infrared (FTIR) spectra under simulated physiological conditions. The binding characteristics of DHA/ART and BSA were determined by fluorescence emission and resonance light scattering (RLS) spectra. The quenching mechanism between BSA and DHA/ART is static. The binding constants and binding sites of DHA/ART–BSA systems were calculated at different temperatures (293, 298, 304 and 310 K). According to Förster non‐radiative energy transfer theory, the binding distance of BSA to DHA/ART was calculated to be 1.54/1.65 nm. The effect of DHA/ART on the secondary structure of BSA was analyzed using UV/vis absorption, FTIR, synchronous fluorescence and 3D fluorescence spectra. In addition, the effects of common ions on the binding constants of BSA–DHA and BSA–ART systems were also discussed. Copyright © 2014 John Wiley & Sons, Ltd.     

超声波清洗器对大鼠心电图的影响

目的考察超声波清洗器对大鼠的影响。方法用RM6240多道生理信号采集处理系统,采用标准肢体二导联法测量正常状态和超声波清洗器工作时麻醉大鼠的心电图。结果超声波清洗器打开后2 min、15 min和30 min时,麻醉大鼠的心率显著增加,心电图的QRS波群间期显著缩短。而超声波清洗器停止工作后30 min时,大鼠心率和QRS波群间期基本恢复至正常。结论超声波清洗器工作时对大鼠心脏可产生一过性的不良影响。因此,在动物饲养或实验区内,超声波清洗器的放置应远离大鼠饲养室。     

In vitro studies on active calcium absorption from ovine rumen

From various in vivo and in vitro studies it has been shown that the rumen represents a significant site of Ca²⁺ absorption in sheep and goats. It was the aim of the present study to further characterize the underlying mechanisms. Unidirectional flux rates of Ca²⁺ across rumen wall epithelia of sheep were measured in vitro by applying the Ussing-chamber technique in the absence of electrochemical gradients. Under these conditions, significant Ca²⁺ net flux rates (J_net) clearly indicate the presence of active mechanisms for Ca²⁺ transport. Short chain fatty acids (SCFAs) caused highest stimulation of Ca²⁺ J_net (6.3 ± 1.9 nmol · cm⁻² · h⁻¹) when used as a mixture of acetate, proprionate and butyrate in physiological proportions (36, 15, 9 mmol · l⁻¹, respectively). The effect of 30 mmol · l⁻¹ butyrate (3.2 ± 0.6 nmol · cm⁻² · h⁻¹) was higher than respective amounts of propionate and acetate (0.6 ± 0.8 nmol · cm⁻² · h⁻¹ and 0.9 ± 0.8 nmol · cm⁻² · h⁻¹, respectively). Eliminating SCFAs resulted in Ca²⁺ J_net of 0.4 ± 1.1 nmol ^. cm^−2 .h⁻¹. Addition of Ca channel blocker verapamil (mucosal 1 mmol · l⁻¹) had no significant effect on SCFA-stimulated J_net of Ca²⁺, whereas application of Na⁺/H⁺ inhibitor amiloride (mucosal 1 mmol · l⁻¹) further enhanced the Ca²⁺ J_net by >65%. The Ca²⁺-pump inhibitor vanadate had no significant effect on J_net of Ca²⁺. Dietary Ca depletion enhanced calcitriol plasma concentrations but had no effect on active Ca²⁺ absorption across the rumen wall of sheep. In addition, no effect on active Ca²⁺ absorption could be observed during early lactation. In conclusion, there is clear evidence for the rumen as a main site for active Ca²⁺ absorption in sheep. Our results suggest the presence of a Ca²⁺/H⁺ exchange mechanism in the apical membrane of rumen epithelial cells which depends on SCFA absorption and which does not seem to be under the control of calcitriol. Basolateral Ca²⁺ extrusion occurs independently from Ca²⁺-pump activity and may be accomplished via Na⁺/Ca²⁺ exchange. Accepted: 29 June 1999     

医学超声治疗原理及其临床应用研究

本文从超声波对人体特有的生物效应出发,用生物医学工程的观点,阐述了现代超声治疗技术基本原理及最新临床应用成果,特别是在超声外科治疗技术中的最新发展,展望了超声治疗技术的应用发展前景。     

Experimental and theoretical studies of cadmium ions absorption by a new reduced recombinant defensin

Heavy metal pollutants such as Cd, Hg, Pb, As, and Se are considered as both a global problem and a growing threat to the humanity. Being strongly poisonous to the metal-sensitive enzymes and leading to the growth inhibition and death of organisms, these metals have a toxic impact on the plants and animals. Inducing the metal-binding cysteine-rich peptides such as metallothioneins, phytochelatins, and defensins, higher organisms like plants and animals usually react to the heavy metal stress. In this study, a recombinant defensin protein was expressed in bean and its ability in the cadmium absorption was determined. Experimental studies revealed that this protein was able to absorb cadmium ions in reduced form more than oxide one. Molecular dynamics simulations were carried out in order to evaluation of experimental studies, using a model of Cd²⁺ or Na⁺ and Cl^– ions enclosed in a fully hydrated simulation box with the recombinant defensin. The theoretical results also suggested that the reduced recombinant defensin was more powerful in the absorption of Cd²⁺ than its oxide form. The present study is the first report of Cd²⁺ absorption potential of this new reduced recombinant defensin. The results unraveled that this recombinant defensin can be adopted as a molecular switch in the cadmium pollution of the environment and also the important role of sulfur groups in the absorption of cadmium ions.     

Ultracentrifugation studies on the dissociation of chemically modified catalases

The dissociation of a series of bovine catalases, in which a proportion of the carboxylic acid groups of glutamic and aspartic acids have been chemically modified by coupling with glycine methyl ester (GME) or ethylenediamine (ED), has been investigated by sedimentation rate and equilibrium methods. Sedimentation equilibrium measurements on GME derivatives have been analysed in terms of a monomer-dimer-trimer- tetramer model. The results show that the association of monomeric (M₁) catalase subunits is consistent with the equilibria 4M₁?2M₂?M₄. The Gibbs energies of association at 284K of the monomeric subunit to dimes (M₂) and tetramers (M₄) were found to be in the range ? 28 to ? 30 kJ mol^?1 and ? 91 to ? 97 kJ mol ^?1, respectively. The Gibbs energy for association of dimer to tetramer is in the range ? 32 to ? 34 kJ mol^?1. Chemical modification of bovine catalase markedly increases its susceptibility to dissociation by sodium n-dodecyl sulphate (SDS) and sedimentation rate measurements suggest that the initial event on addition of SDS is the dissociation of the whole molecule to half-molecules     

Stereospecific interactions between histidine and monoclonal antibodies

Histidine is a frequently used buffer in the final formulation of many commercialized monoclonal antibodies (mAbs), with histidine helping to stabilize the antibody during storage in addition to its buffering function. The objective of this study was to examine the stereospecificity of any histidine-antibody interactions using a combination of experimental studies and molecular dynamics simulations. Isothermal titration calorimetry provided evidence of weak stereospecific interactions, with the antibody showing approximately two to four additional interaction sites for d - versus l -histidine. The greater interactions with d -histidine were confirmed by measurements of the net protein charge using electrophoretic light scattering. The reduction in the net negative charge of the antibody in d -histidine led to significantly different behavior during diafiltration due to Donnan exclusion effects. Molecular dynamics simulations corroborated the presence of additional d -histidine interaction sites. These results provide the first demonstration of weak stereospecific interactions between l - and d -histidine and a mAb and the implications of these interactions for antibody formulation.