Application of an optimized marker amplification protocol in immunohistochemistry — a valuable tool for visualizing antigenic sites of low signal strength or sparse distribution

Amplification of immunohistochemical markers received considerable attention during the 1980s and 1990s. The amplification approach was largely abandoned following the development of antigen retrieval and reporter amplification techniques, because the latter were incorporated more easily into high throughput automated procedures in industrial and diagnostic laboratories. There remain, however, a number of instances where marker amplification still has much to offer. Consequently, we examined experimentally the utility of an optimized marker amplification technique in diagnostically relevant tissue where either the original signal strength was low or positive sites were visible, but sparsely distributed. Marker amplification in the former case not only improved the visibility of existing positive sites, but also revealed additional sites that previously were undetectable. In the latter case, positive sites were rendered more intense and therefore more easily seen during low magnification examination of large areas of tissue.     

Solubilization of an Adenosine Uptake Site in Brain

Procedures are described for the solubilization of adenosine uptake sites in guinea pig and rat brain tissue. Using [3H]nitrobenzylthioinosine [( 3H]NBI) the solubilized site is characterized both kinetically and pharmacologically. The binding is dependent on protein concentration and is saturable, reversible, specific, and high affinity in nature. The KD and Bmax of guinea pig extracts are 0.13 +/- 0.02 nM and 133 +/- 18 fmol/mg protein, respectively, with linear Scatchard plots obtained routinely. Similar kinetic parameters are observed in rat brain. Adenosine uptake inhibitors are the most potent inhibitors of [3H]NBI binding with the following order of potency, dilazep greater than hexobendine greater than dipyridamole. Adenosine receptor ligands are much less potent inhibitors of binding, and caffeine is without effect. The solubilized adenosine uptake site is, therefore, shown to have virtually identical properties to the native membrane site. The binding of the adenosine A1 receptor agonist [3H]cyclohexyladenosine [( 3H]CHA) to the solubilized brain extract was also studied and compared with that of [3H]NBI. In contrast to the [3H]NBI binding site [3H]CHA binds to two apparent populations of adenosine receptor, a high-affinity site with a KD of 0.32 +/- 0.06 nM and a Bmax of 105 +/- 30 fmol/mg protein and a lower-affinity site with a KD of 5.50 +/- 0.52 nM and Bmax of 300 +/- 55 fmol/mg protein. The pharmacology of the [3H]CHA binding site is consistent with that of the adenosine receptor and quite distinct from that of the uptake [( 3H]NBI binding) site. Therefore, we show that the adenosine uptake site can be solubilized and that it retains both its binding and pharmacologic properties in the solubilized state.     

The effect of different blood sampling sites and analyses on the relationship~ between exercise intensity and 4.0 mmol ·1? blood lactate concentration

The aim of the study was to examine whether the difference in lactate concentration in different blood fractions is of practical importance when using blood lactate as a test variable of aerobic endurance capacity. Ten male firefighters performed submaximally graded exercise on a cycle ergometer for 20-25 min. Venous and capillary blood samples were taken every 5 min for determination of haematocrit and lactate concentrations in plasma, venous and capillary blood. At the same time, expired air was collected in Douglas bags for determination of the oxygen consumption. A lactate concentration of 4.0 mmol.l-1 was used as the reference value to compare the oxygen consumption and exercise intensity when different types of blood specimen and sampling sites were used for lactate analysis. At this concentration the exercise intensity was 17% lower (P less than 0.01) when plasma lactate was compared to venous blood lactate, and 12% lower (P less than 0.05) when capillary blood lactate was used. Similar discrepancies were seen in oxygen consumption. The results illustrated the importance of standardizing sampling and handling of blood specimens for lactate determination to enable direct comparisons to be made among results obtained in different studies.     

Discrimination of Multiple [3H]5-Hydroxytryptamine Binding Sites by the Neuroleptic Spiperone in Rat Brain

Certain neuroleptic drugs, such as spiperone and (+) butaclamol, can discriminate between two populations of [³H]5-hydroxytryptamine ([³H]5-HT) binding sites in rat brain. The butyrophenone neuroleptic spiperone shows the greatest selectivity for these two binding sites, having at least a 3000-fold difference between its dissociation constants (2-12 nM versus 35,000 nM) for the high- and low-affinity sites, respectively. Inhibition of [³H]5-HT binding by spiperone in rat frontal cortex and corpus striatum yields distinctly biphasic inhibition curves with Hill slopes significantly less than unity. Results from nonlinear regression analysis of these inhibition studies were consistent with a two-site model in each brain region. In the frontal cortex the high-affinity neuroleptic sites comprised about 60% of the total [^3/H]5-HT binding sites whereas in the corpus striatum they accounted for only 20% of the sites. Furthermore, saturation studies of [³H]5-HT binding assayed in the absence or presence of 1 μM-spiperone (a concentration that completely blocks the high-affinity site while having minimal activity at the low-affinity site) reveal a parallel shift in the Scatchard plot with no change in the dissociation constant of [³H]5-HT, but a significant decrease (64% in frontal cortex or 28% in corpus striatum) in the number of specific binding sites. These observations are consistent with the existence of at least two populations of [³H]5-HT binding sites having a differential regional distribution in rat brain.     

Exploring the obscure profiles of pharmacological binding sites on voltage-gated sodium channels by BmK neurotoxins

Diverse subtypes of voltage-gated sodium channels (VGSCs) have been found throughout tissues of the brain, muscles and the heart. Neurotoxins extracted from the venom of the Asian scorpion Buthus martensi Karsch (BmK) act as sodium channel-specific modulators and have therefore been widely used to study VGSCs. α-type neurotoxins, named BmK I, BmK αIV and BmK abT, bind to receptor site-3 on VGSCs and can strongly prolong the inactivation phase of VGSCs. In contrast, β-type neurotoxins, named BmK AS, BmK AS-1, BmK IT and BmK IT2, occupy receptor site-4 on VGSCs and can suppress peak currents and hyperpolarize the activation kinetics of sodium channels. Accumulating evidence from binding assays of scorpion neurotoxins on VGSCs, however, indicate that pharmacological sensitivity of VGSC subtypes to different modulators is much more complex than that suggested by the simple α-type and β-type neurotoxin distinction. Exploring the mechanisms of possible dynamic interactions between site 3-/4-specific modulators and region- and/or species-specific subtypes of VGSCs would therefore greatly expand our understanding of the physiological and pharmacological properties of diverse VGSCs. In this review, we discuss the pharmacological and structural diversity of VGSCs as revealed by studies exploring the binding properties and cross-competitive binding of site 3- or site 4-specific modulators in VGSC subtypes in synaptosomes from distinct tissues of diverse species.     

Factors influencing the habitat choice of pangolins (Manis spp.) in low land of Nepal

Pangolins in the genus Manis are nocturnal, burrowing, insectivorous mammals listed as Critically Endangered or Endangered by the International Union for Conservation of Nature. Two species of pangolins are found in Nepal: the Chinese pangolin (Manis pentadactyla) and Indian pangolin (Manis crassicaudata). Despite having high conservation priority, little attention has been given to conservation interventions of both species of pangolins found in the Terai region (low land) of Nepal. The present study assesses habitat use and factors affecting the habitat choice of pangolins in low land (Terai), Nepal, focusing on Amritdharapani Community Forest of Chitwan district. Pangolin burrows were used as the indirect signs, and opportunistic sampling method was used to record the burrows. After the identification of all occurrence sites (burrows) in the field, random points were generated excluding the points where burrows were recorded for sampling of nonoccurrence sites. A total of thirty‐nine burrows were observed at elevations ranging from 301 to 413 masl. Burrows were frequently associated with northwest aspects, gentle slope (15°–20°), moderate canopy cover (51%–75%), red‐colored soil, and acidic soils with pH 6.5–7. The burrows were most common in areas with weak human disturbance (i.e., 1,500–1,700 m from settlements), 800–1,200 m from roads, and within 300 m from a water source and within 20 m from the nearest termitarium. Distance to settlement, distance to road, soil pH, and canopy cover were found to affect the habitat choice of pangolins in the study area.     

塔克拉玛干沙漠不同区域柽柳沙包土壤盐分分布特征及其影响因素

柽柳(Tamarix chinensis)沙包是塔克拉玛干沙漠特殊的生物地貌景观, 对维持区域生态环境的稳定具有极其重要的作用。该研究采用野外调查与室内分析相结合的方法, 选取且末、阿拉尔、策勒、塔中4个典型区域的柽柳沙包为研究对象, 对柽柳沙包0-500 cm土壤垂直剖面进行采样, 测定土壤pH值、枯落物含量、电导率及HCO₃ ^-、Cl ^-、SO₄ ^2-、Ca ²⁺、Mg ²⁺、K ⁺、Na ⁺含量, 分析柽柳沙包中土壤盐分的空间变化规律及其影响因素。结果表明: 1)从且末、阿拉尔、策勒到塔中, 土壤pH值总体呈升高趋势, 土壤电导率及Na ⁺、Ca ²⁺、Mg ²⁺、SO₄ ^2-含量总体呈降低趋势, K ⁺、Cl ^-、HCO₃ ^-含量没有明显的变化规律。2)盐分在4个样区的垂直分布主要表现为: 且末和策勒样区柽柳沙包的土壤盐分呈表层聚集现象; 阿拉尔和塔中样区柽柳沙包的土壤盐分呈深层聚集现象。随着土层深度的增加, 土壤pH值总体呈升高的趋势, 土壤枯落物含量总体呈降低趋势; 土壤电导率在且末和策勒样区总体呈降低趋势, 阿拉尔样区呈先降低后升高再降低的变化趋势, 而塔中样区呈先升高后降低再升高的变化趋势。3)根据相关性分析和主成分分析, 且末样区土壤枯落物含量、SO₄ ^2-、Na ⁺、K ⁺为影响土壤盐分含量的主要因子, 且土壤盐分以硫酸盐为主; 阿拉尔样区影响土壤盐分组成的主要因子为Cl ^-、Na ⁺; 策勒样区为Cl ^-、K ⁺、Na ⁺; 塔中样区为Cl ^-、Na ⁺、Ca ²⁺、SO₄ ^2-, 且土壤盐分均以氯化物为主。综合分析表明, 不同区域柽柳沙包中土壤盐分存在空间变异性, 柽柳沙包土壤盐分的变化与干旱沙漠地区强烈的蒸发作用、地表风蚀强度、地下水埋深、土壤中枯落物及柽柳的生物积盐效应等因素密切相关, 是影响不同区域土壤盐分分布的关键因子。     

Two uncompetitive, activated, and transport sites of the Na+/H+ exchanger for pH regulation in perfused rat kidney

The purpose of this study is to assess the effect of an apparent alteration in intracellular pH and the effect of amiloride on the activity of the Na+/H+ antiporter in perfused rat kidney. Rat kidney-Na+ retention was determined using tracer 22Na in perfusate composed of HCl-glycine buffer (pH 3.80 to pH 5.92) or NH4OH-glycine buffer (pH 6.22-7.95) containing Na+ to match physiologic concentrations. Plotting renal Na+ retention for 10 min versus pH in absence of amiloride showed two classical uncompetitive activator curves for H+, one curve from pH 4.19 to 5.10 and another from pH 6.22 to 7.95. H+ acts as an uncompetitive reversible binding substrate with the receptor triggering activation of the exchanger already sequestered with Na+, thus yielding two Ka values for the exchanger suggesting non-first order kinetics. Using an equation derived for uncompetitive-activation binding of Nao+ and Hi+, plotting [mM Na+ mg protein-1 10 min-1]-1 versus [H+], two linear plots are observed on Cartesian coordinates with abscissa intersecting at 47 +/- 1 microM, pKa = 4.32 +/- 0.02 (pH 4.19-5.10) and 4.21 +/- 0.02 microM, pKa = 5.38 +/- 0.01 (pH 6.22-7.95), respectively. Perfusing buffer containing 2 mM amiloride, completely inactivated the antiporter showing stronger inhibition between pH 3.80 and 5.92. Results suggest the presence of two uncompetitive binding sites for H+ with the Na+/H+ exchanger. One is a high affinity binding site at physiological intracellular apparent pH, and another is a low affinity binding site at ischaemic apparent pH, implying the existence of two titration sites for intracellular pH regulation.     

东亚-澳大利西亚候鸟迁飞区中杓鹬 的迁徙追踪

追踪候鸟的迁徙活动是全面认识其生活史年周期的重要途径。中杓鹬(Numeniusphaeopus)在全球广泛分布,但在东亚-澳大利西亚候鸟迁飞区的迁徙活动一直缺乏追踪研究。2018年2月,在澳大利亚西北部的布鲁姆为捕捉到的中杓鹬成鸟佩戴平台发射终端或全球定位系统-全球移动通讯系统追踪器,以确定其迁徙日程、迁徙路线以及迁徙停歇地和繁殖地的地理位置。我们从成功追踪的7只个体获取了6 378条精度高于1 km的位点数据。分析结果表明,在春季,中杓鹬的迁徙时长为(36±4)d,其间在1～3个迁徙停歇地的停留日期为(23±2)d,从越冬地到繁殖地的迁徙距离为(9 795±346)km(n=7)。追踪的中杓鹬在俄罗斯东部和中部区域繁殖,不同个体的繁殖地纬度相近而经度范围较广。在秋季,中杓鹬的迁徙时长为(90±27)d,相比春季迁徙时长更长;其间,在2～4个迁徙停歇地停留(79±29)d,从繁殖地到越冬地的迁徙距离为(10 101±520)km(n=5)。无论在春季还是秋季迁徙,迁徙停歇地广泛分布于东亚、东南亚沿海及内陆区域。大部分个体春季和秋季的迁徙路线相近,成功追踪的个体均在秋季返回了上一年的越冬地,这表明中杓鹬对越冬地具有很高的忠诚度。     

Cleavage of the Carboxyl-Terminus of LEACS2, a Tomato 1-Aminocyclopropane-1-Carboxylic Acid Synthase Isomer, by a 64-kDa Tomato Metalloprotease Produces a Truncated but Active Enzyme

1-Aminocyclopropane-1-carboxylic acid （ACC） synthase （ACS） is the principal enzyme in phytohormone ethylene biosynthesis. Previous studies have shown that the hypervariable C-terminus of ACS is proteolytically processed in vivo. However, the protease responsible for this has not yet been identified. In the present study, we investigated the processing of the 55-kDa full-length tomato ACS （LeACS2） into 52-, 50- and 49-kDa truncated isoforms in ripening tomato （Lycopersicon esculentum Mill. cv. Cooperation 903） fruit using the sodium dodecyl sulfate-boiling method. Meanwhile, an LeACS2-processing protease was purified via multi-step column chromatography from tomato fruit. Subsequent biochemical analysis of the 64-kDa purified protease revealed that it is a metalloprotease active at multiple cleavage sites within the hypervariable C-terminus of LeACS2. N-terminal sequencing and matrix-assisted laser desorption/ionization time-of-flight analysis indicated that the LeACS2-processing metalloprotease cleaves at the C-terminal sites Lys^438, Glu^447, Lys^448, Asn^456, Ser^460, Ser^462, Lys^463, and Leu^474, but does not cleave the N- terminus of LeACS2. Four C-terminus-deleted （26-50 amino acids） LeACS2 fusion proteins were overproduced and subjected to proteolysis by this metalloprotease to identify the multiple cleavage sites located on the N-terminal side of the phosphorylation site Ser^460. The results indisputably confirmed the presence of cleavage sites within the region between the α-helix domain （H14） and Ser^460 for this metalloprotease. Furthermore, the resulting C-terminally truncated LeACS2 isoforms were active enzymatically. Because this protease could produce LeACS2 isoforms in vitro similar to those detected in vivo, it is proposed that this metalloprotease may be involved in the proteolysis of LeACS2 in vivo.