新型蛋白质修饰剂的合成及修饰牛血红蛋白的初步研究

以L-谷氨酸和己二酸为原料合成了一种新型的四官能团蛋白质修饰剂,并用核磁共振和红外光谱对其结构进行了表征。然后以其为修饰剂,对牛血红蛋白的化学修饰进行了初步的研究,并通过高效液相色谱、聚丙烯酰胺凝胶电泳和血氧分析仪对交联牛血红蛋白的分子量和携氧性能进行了表征。结果表明,该修饰剂可以使牛血红蛋白同时在分子内和分子间发生化学交联,并较好地保持携氧能力（P50:21.7mmHg,Hill系数:2.01）,因此在众多用于开发人工血液代用品的化学修饰剂中该修饰剂具有良好的应用前景。     

功能磁共振成像北大核心CSCD

功能磁共振成像（functional MRI）是一项通过检测大脑神经元活动引起的局部血氧浓度变化来探测脑功能情况的影像技术。大脑神经元活动引起的能量同时伴随着氧的代谢,血液中的含氧血红蛋白由此转为脱氧血红蛋白。一般情况下,脑功能活动增加而引起的氧耗要小于由脑血流供应带来的氧增加,引起该区域最终的血氧浓度反而升高。含氧血红蛋白和脱氧血红蛋白对磁场的影响不同,造成磁共振信号的相应变化。同一区域的血氧浓度高则磁共振信号越强。     

牛血红蛋白PLGA纳米粒的制备及其体外功能的研究

目的:利用poly(lactide-co-glycolide)(PLGA)和poly(styrene-co-4-styrene-sulfonate)(PSS)制备带负电荷的牛血红蛋白纳米粒,并对其载氧性能和体外性能进行评价.方法:采用溶剂蒸发法制备出空白的PLGA-PSS的空白纳米粒,通过改变pH值,吸附牛血红蛋白,从而制备出牛血红蛋白PLGA纳米粒.通过TEM、粒径、Zeta电位、包裹率和载药量、体外释放及其携氧能力对该纳米粒进行了综合分析.结果:Hb-PLGA-PSS-NPsTEM电镜下呈类球形,平均粒径为226.8±23.4nm,Zeta电位为-68.62 mV,优化条件后最大包裹率约为99.3%,载药量约为28.6%,在37℃,pH7.4的PBS溶液中释放缓慢.通过对Hb结构的分析表明此工艺未对蛋白的结构造成影响,体外携氧实验测量了P50(29mmHg)和Hill系数(2.036),结果说明该Hb纳米微囊具有良好携氧功能.结论:成功制备了一种Hb-PLGA-PSS-NPs纳米粒,稳定性好,具有很好的携氧能力.     

血红蛋白的化学修饰与血液代用品

血液代用品系指具有携氧扩容功效的溶液,包括：氟碳化合物、脂质体包封血红蛋白（hemoglobin,Hb)、微囊化血红蛋白和无细胞基质血红蛋白（化学修饰Ｈb、基因重组Ｈb、化学合成Ｈb和转基因动物人Ｈb）。通过化学修饰稳定血红蛋白结构以避免其肾毒性,并延长循环半寿期是利用无基质血红蛋白制备血液代用品的主要研究内容,通常采用交联、聚合、偶联等方法完成。     

红景天虫草复方可促进骨骼肌和大脑组织氧合作用

为了探讨补充红景天与冬虫夏草复方对短期高原训练后骨骼肌与大脑组织氧合作用影响,本研究招募22位男性田径运动员,按照最大摄氧量配对分组成安慰剂组(placebo,P)和红景天与冬虫夏草复方组(rhodiola crenulata with cordyceps sinensis,RC)。通过在受试者进行高原训练前2周开始,使其每天摄入安慰剂和红景天与冬虫夏草复方,连续补充至2周高原训练结束为止,共计补充4周,RC组每日补充量为2 000 mg。并在高原训练前和2周训练后隔天,测量受试者在海平面高度安静状态时骨骼肌与大脑组织总血红蛋白浓度、携氧血红蛋白浓度、脱氧血红蛋白浓度和氧饱和度百分比后发现,P组大脑组织总血红蛋白浓度、携氧血红蛋白浓度、脱氧血红蛋白浓度在2周高原训练后明显低于训练前;RC组能正常维持大脑组织氧气供应;且在2周高原训练后,P与RC两组安静状态骨骼肌总血红蛋白浓度、携氧血红蛋白浓度及氧饱和度百分比明显高于训练前。这说明可以通过补充红景天与冬虫夏草复方来预防高原训练后大脑组织总血红蛋白浓度、携氧血红蛋白与脱氧血红蛋白浓度下降的情况。     

虎纹捕鸟蛛毒素-X的化学合成及其鉴定

应用芴甲氧羰基（Fmoc）固相方法化学合成了虎纹捕鸟蛛毒素-X（huwentoxin-X,HWTX-X）,并在含5mmol／LGSH和0．5mmol／LGSSG的0．1mol／L Tris-HCl溶液（pH8．0）中氧化复性：复性产物与天然多肽具有相同的分子质量,在反相HPLC上共洗脱,其一维核磁共振谱规则分散,表明合成多肽形成了3对二硫键和稳定的空间结构,并与天然多肽相同：此合成的多肽能专一性抑制大鼠背根神经节细胞上N-型电压敏感钙离子通道．其IC50约40nmol／L. HWTX-X的成功合成和鉴定为进一步研究HWTX-X的结构与功能关系、药理学性质以及新型镇痛药物研发奠定了基础．     

近红外光谱技术在神经外科患者脑氧和血流动力学监测中的应用研究进展

神经外科患者,尤其是脑血流自动调节功能受损的重症患者,脑氧饱和度是反应患者脑组织氧代谢情况的重要指标,实时、准确的脑氧饱和度监测方法对于指导选择有效的治疗措施和判断患者预后具有重要价值。基于血红蛋白不同氧合状态,即氧合血红蛋白(oxyhemoglobin,Hb O2),还原血红蛋白(deoxygenated hemoglobin,Hb)具有的差异性分子光谱,近红外光谱技术near infrared spectroscopy,NIRS)可监测人体局部组织氧饱和度。由于近红外射线能穿透颅骨直接获得脑组织内平均氧饱和度的特性,可协助临床实现无创持续监测脑氧饱和度的目的,近年来该技术在神经外科领域的应用研究获得了迅速发展,在颅脑创伤和其它神经外科疾病的应用研究中均取得了显著的进展,本文将对最新研究结果及其意义和未来发展方向进行综述。     

营养状况与老年稳定期慢性阻塞性肺疾病患者康复的关系

目的：探讨和研究营养状况与老年稳定期慢性阻塞性肺疾病患者康复之间的关系。方法：选取内蒙古医科大学附属医院2013年2月—2017年12月收治的慢性阻塞性肺疾病患者96例,根据微型营养评定法分为营养良好组31例、营养不良危险组30例和营养不良组35例,分析和比较3组肺功能、血气分析以及氧代谢各指标水平。结果：营养良好组患者肺活量（FVC）、用力呼气中期流速（MMF）、第1秒用力呼吸容积所占百分比（FEV1%）、大通气功能（MVV）、氧分压（PaO2）、血氧饱和度（SaO2）以及静脉血氧饱和度（SvO2）、摄氧率（VO2）、氧合指数（CaO2）等指标均明显高于营养不良危险组和营养不良组,而营养不良危险组上述指标明显高于营养不良组（P＜0.05或P<0.01）,二氧化碳分压（PaCO2）和氧摄取率（ERO2）营养良好组明显低于其他两组,而营养不良危险组上述指标明显低于营养不良组（P<0.05或P＜0.01）,3组pH无显著差异（P>0.05）。结论：老年稳定期慢性阻塞性肺疾病患者的营养状况与肺功能、氧代谢和血气分析指标具有明显相关性,临床上在治疗老年稳定期COPD时通过改善其营养状况可有效促进患者康复。     

急性低氧对耐力运动员无氧代谢阈值的影响

通过观察急性低氧对耐力运动员无氧代谢阈值的影响,探讨了急性低氧对耐力运动员体力活动能力的影响,并分析与上述影响强弱有关的若干可能因素。18名青年耐力运动员分别吸入21％O_2(常氧对照)和12.8％O_2(急性低氧),进行逐级递增体力负荷运动,直至最大耐受量。观察运动期间每分通气量、氧耗量、二氧化碳排出量、动脉血氧饱和度、心率和氧脉搏,并测定无氧代谢阈值。结果表明,急性低氧使耐力运动员的无氧代谢阈值明显降低。各个体无氧代谢阈值的降低程度分别与该个体动脉血氧饱和度以及氧脉搏的降低程度呈正相关。急性低氧下无氧代谢阈值降低提示了体力活动能力的削弱,而低氧下心肺代偿功能较弱以及动脉血氧饱和度较低者,其体力活动能力的削弱较明显。     

血红蛋白携氧-释氧动力学研究

本文研究了鸡、家兔、鲤鱼、蟾蜍4种实验动物血红蛋白(hemoglobin,Hb)携氧-释氧动力学过程,初步建立Hb携氧-释氧动力学研究方法,并探讨Hb携氧-释氧动力学过程与动物生存环境之间的关系.结果显示:4种动物Hb携氧动力学曲线均呈"S"形曲线特征,与传统的Hb氧解离曲线(oxygen dissociation curve,ODC)相似;同时不同动物Hb携氧-释氧动力学曲线也有各自特点,如鸡Hb释氧时间长达(1 411±6)S;在Hb携氧.释氧曲线I阶段,鲤鱼上升斜率远大于家兔等.提示Hb携氧-释氧动力学曲线可反映不同动物Hb携氧效率的差异.与传统ODC参数P50相对应,由动力学曲线可得到Hb携氧动力学参数T50°T50是Hb达到50%氧饱和度所需时间,可直观反映Hb携氧效率的差异.4种实验动物Hb均有较稳定的T50,从大到小依次为:鸡、家兔、鲤鱼和蟾蜍.对Hb携氧动力学曲线与ODC综合分析,可得到Hb携氧效能参数E50,表示Hb达到50%氧饱和度所用时间与环境氧分压之间的关系,即E50(50% Sat,Xeo2,yr).E50有可能成为全面评价Hb携氧效能的综合指标.     

Bone morphogenetic protein-2 and transforming growth factor-β2 interact to modulate human bone marrow stromal cell proliferation and differentiation

Osteoprogenitor cells in the human bone marrow stroma can be induced to differentiate into osteoblasts under stimulation with hormonal and local factors. We previously showed that human bone marrow stromal (HBMS) cells respond to dexamethasone and vitamin D by expressing several osteoblastic markers. In this study, we investigated the effects and interactions of local factors (BMP-2 and TGF-β₂) on HBMS cell proliferation and differentiation in short-term and long-term cultures. We found that rhTGF-β₂ increased DNA content and stimulated type I collagen synthesis, but inhibited ALP activity and mRNA levels, osteocalcin production, and mineralization of the matrix formed by HBMS cells. In contrast, rhBMP-2 increased ALP activity and mRNA levels, osteocalcin levels and calcium deposition in the extracellular matrix without affecting type I collagen synthesis and mRNA levels, showing that rhBMP-2 and rhTGF-β₂ regulate differentially HBMS cells. Co-treatment with rhBMP-2 and rhTGF-β₂ led to intermediate effects on HBMS cell proliferation and differentiation markers. rhTGF-β₂ attenuated the stimulatory effect of rhBMP-2 on osteocalcin levels, and ALP activity and mRNA levels, whereas rhBMP-2 reduced the rhTGF-β₂-enhanced DNA synthesis and type I collagen synthesis. We also investigated the effects of sequential treatments with rhBMP-2 and rhTGF-β₂ on HBMS cell differentiation in long-term culture. A transient (9 days) treatment with rhBMP-2 abolished the rhTGF-β₂ response of HBMS cells on ALP activity. In contrast, a transient (10 days) treatment with rhTGF-β₂ did not influence the subsequent rhBMP-2 action on HBMS cell differentiation. The data show that TGF-β₂ acts by increasing HBMS cell proliferation and type I collagen synthesis whereas BMP-2 acts by promoting HBMS cell differentiation. These observations suggest that TGF-β₂ and BMP-2 may act in a sequential manner at different stages to promote human bone marrow stromal cell differentiation towards the osteoblast phenotype. J. Cell. Biochem. 68:411–426, 1998. © 1998 Wiley-Liss, Inc.     

Hemoglobin from a deep-sea hydrothermal-vent copepod

Deep-sea hydrothermal-vent fauna live in a highly variable environment where oxygen levels can be very low, and carbon dioxide and sulfide can reach high concentrations (1). These conditions are harsh for most aerobic metazoans, yet copepods can be abundant at hydrothermal vents. Here we report the structure and functional properties of hemoglobin extracted from the copepod Benthoxynus spiculifer, which was found in large numbers in a paralvinellid/gastropod community collection made during a cruise to the Juan de Fuca Ridge in 1998. Although hemoglobin has been reported in some littoral copepods (2), this is the first study of the structure and functional properties of copepod hemoglobin. Hemoglobin represents about 60% of the total soluble proteins extracted from B. spiculifer, and although it imparts a red color to the copepod, it does not provide a significant storage pool of oxygen. It is a 208-kDa protein, composed of 14 globin chains--7 of 14.3 kDa and 7 of 15.2 kDa. The hemoglobin has a very high and temperature-sensitive oxygen affinity, with no cooperativity or Bohr effect. These properties are adaptive for an animal living in a low-oxygen environment in which the primary function of the hemoglobin is most likely oxygen acquisition to support aerobic respiration.     

Hemoglobin Tilburg: alpha 2-beta 2 73 (E 17) Asp----Gly. A new hemoglobin with reduced oxygen affinity

A new hemoglobin variant has been found in a Dutch Caucasian girl and detected also in members of three generations of her family. This variant is characterized by the substitution of an aspartic acid at position 73 (E 17) of the beta-chain with a glycine residue. Hemoglobin Tilburg makes up to 42% of the total hemoglobin in the blood of the proposita, it is stable at the isopropanol test, and not associated with significant hematological abnormalities in heterozygous carriers. The oxygen dissociation curve of the purified variant, carried out at different pH values, shows a definite reduction of the affinity for oxygen and a normal alkaline Bohr effect. Three more hemoglobins with a single amino acid substitution at the same site have been previously described: Hb Korle-Bu (Asp----Asn), Hb Mobile (Asp----Val) and Hb Vancouver (Asp----Tyr). In all these proteins the affinity for oxygen is lowered to an extent which is variable and characteristic of each mutant. In this paper we discuss the possible mechanism responsible for the abnormal behaviour of hemoglobins substituted at beta 73.     

Intrinsic oxygen affinity of hemoglobins: the hemoglobin of bisons (Bison bonasus,Bovidae)

The hemoglobin from a European Bison (Bison bonasus) was analysed and the complete primary structures of the alpha I-, alpha II-and beta-chains have been determined. The alpha I- and alpha II-chains differ only at position alpha 19 (Asp----Gly). The beta-chains are homogeneous. The sequences are compared with the globin chains of Bison bison and bovine and the polymorphism of the alpha-chains is discussed. On the basis of the primary structure it may be concluded that the hemoglobin of Bison bonasus belongs to the group of hemoglobins with intrinsically low oxygen affinity.     

Hemoglobin tilburg: α2-ß2 73 (E 17) Asp→Gly: A new hemoglobin with reduced oxygen affinity

A new hemoglobin variant has been found in a Dutch Caucasian girl and detected also in members of three generations of her family. This variant is characterized by the substitution of an aspartic acid at position 73 (E 17) of the ß-chain with a glycine residue. Hemoglobin Tilburg makes up to 42% of the total hemoglobin in the blood of the proposita, it is stable at the isopropanol test, and not associated with significant hematological abnormalities in heterozygous carriers. The oxygen dissociation curve of the purified variant, carried out at different pH values, shows a definite reduction of the affinity for oxygen and a normal alkaline Bohr effect. Three more hemoglobins with a single amino acid substitution at the same site have been previously described: Hb Korle-Bu (Asp→Asn), Hb Mobile (Asp→Val) and Hb Vancouver (Asp→Tyr). In all these proteins the affinity for oxygen is lowered to an extent which is variable and characteristic of each mutant. In this paper we discuss the possible mechanism responsible for the abnormal behaviour of hemoglobins substituted at ß 73.     

Reverse Engineering the Cooperative Machinery of Human Hemoglobin

Hemoglobin transports molecular oxygen from the lungs to all human tissues for cellular respiration. Its α₂β₂ tetrameric assembly undergoes cooperative binding and releasing of oxygen for superior efficiency and responsiveness. Over past decades, hundreds of hemoglobin structures were determined under a wide range of conditions for investigation of molecular mechanism of cooperativity. Based on a joint analysis of hemoglobin structures in the Protein Data Bank (Ren, companion article), here I present a reverse engineering approach to elucidate how two subunits within each dimer reciprocate identical motions that achieves intradimer cooperativity, how ligand-induced structural signals from two subunits are integrated to drive quaternary rotation, and how the structural environment at the oxygen binding sites alter their binding affinity. This mechanical model reveals the intricate design that achieves the cooperative mechanism and has previously been masked by inconsistent structural fluctuations. A number of competing theories on hemoglobin cooperativity and broader protein allostery are reconciled and unified.     

Interaction between Protein Subunits from Model Studies

A molecular model of hemoglobin was constructed which made it possible to visualize the relation between various amino acid residues in the molecule. The model indicates that electrostatic forces might play a significant role in holding the subunits of hemoglobin together. This would explain why myoglobin does not form a tetramer while four β-chains, which are structurally similar to myoglobin, do assemble into a hemoglobin H molecule. Also, as far as the primary structures of hemoglobin chains of various species are known, the proposed ionic links between subunits are consistent with the fact that mammalian hemoglobins form stable tetramers while the peptide chains of lamprey hemoglobin are only weakly associated. The different behavior of hemoglobin H and of normal hemoglobin upon oxygen uptake is briefly discussed in terms of allosteric effects.     

A reaction cell for simultaneous measurements of fluorescence and absorption in a hemoglobin solution at various oxygen pressures

An apparatus was constructed to carry out measurements of fluorescence, optical absorption and oxygen partial pressure in a hemoglobin or other solution simultaneously, and its performance was examined. This apparatus has a rhombiform optical cell in place of the usual square optical cell used in commercially available spectrofluorometers. Fluorescence emitted at the region near the cell surface in the solution could be detected satisfactorily and easily even if the solution had strong light absorption bands at both the excitation and the emission wavelengths in the presence of high concentrations of a chromophore. This apparatus was particularly effective for studies on the interactions of a fluorescent allosteric effector with hemoglobin at various degrees of deoxygenation. Consequently, it was proved experimentally that the fluorescence of β-naphthyl triphosphate bound to hemoglobin is completely quenched. Moreover, simultaneous and continuous measurements of the oxygen-binding equilibrium of hemoglobin and the allosteric effector-binding to hemoglobin as a function of oxygen partial pressure could be satisfactorily carried out, and it is confirmed that β-naphthyl triphosphate binds not only to deoxyhemoglobin but also to fully oxygenated hemoglobin and lowers strongly the oxygen affinity of hemoglobin as an allosteric effector.     

Gas transfer system in Alvinella pompejana (Annelida polychaeta, Terebellida): functional properties of intracellular and extracellular hemoglobins

Alvinella pompejana is a tubicolous polychaete that dwells in the hottest part of the hydrothermal vent ecosystem in a highly variable mixture of vent (350 degrees C, anoxic, CO(2)- and sulfide-rich) and deep-sea (2 degrees C, mildly hypoxic) waters. This species has developed distinct-and specifically respiratory-adaptations to this challenging environment. An internal gas exchange system has recently been described, along with the report of an intracellular coelomic hemoglobin, in addition to the previously known extracellular vascular hemoglobin. This article reports the structure of coelomic hemoglobin and the functional properties of both hemoglobins in order to assess possible oxygen transfer. Coelomocytes contain a unique monomeric hemoglobin with a molecular weight of 14,810+/-1.5 Da, as determined by mass spectrometry. The functional properties of both hemoglobins are unexpectedly very similar under the same conditions of pH (6.1-8.2) and temperature (10 degrees -40 degrees C). The oxygen affinity of both proteins is relatively high (P50=0.66 Torr at 20 degrees C and pH 7), which facilitates oxygen uptake from the hypoxic environment. A strong Bohr effect (Phi ranging from -0.8 to -1.0) allows the release of oxygen to acidic tissues. Such similar properties imply a possible bidirectional transfer of oxygen between the two hemoglobins in the perioesophagal pouch, a mechanism that could moderate environmental variations of oxygen concentration and maintain brain oxygenation.     

The hemoglobin of the sea lamprey,Petromyzon marinus

The blood hemoglobin of the sea lamprey presents a curious mixture of primitive and highly specialized properties. Like muscle hemoglobin, it has a molecular weight of about 17,000, and apparently contains a single heme. Its isoelectric point is like that of a typical invertebrate hemoglobin. Its amino acid composition is partly characteristic of invertebrate) partly of vertebrate hemoglobins (Pedersen; Roche and Fontaine). In the present experiments, the oxygen equilibrium curve of this pigment was measured at several pH's. As expected, it is a rectangular hyperbola, the first such function to be observed in a vertebrate blood hemoglobin. Other hemoglobins known to possess this type of oxygen dissociation curve—those of vertebrate muscle, the worm Nippostrongylus, and the bot-fly larva—appear to serve primarily the function of oxygen storage rather than transport. Lamprey hemoglobin on the contrary is an efficient oxygen-transporting agent. It achieves this status by having, unlike muscle hemoglobin, a relatively low oxygen affinity, and a very large Bohr effect. In these properties it rivals the most effective vertebrate blood hemoglobins.