东北虎幼体血清蛋白及胆汁酸的测定与分析

用HITACHI 7600-20全自动生化分析仪对13只东北虎幼体进行了总蛋白、白蛋白、球蛋白、前白蛋白及胆汁酸5项生化指标的测定,实验数据经SPSS软件统计分析.其结果为:总蛋白含量66.06±6.80 g/L,白蛋白含量34.19±2.51 g/L,球蛋白含量31.89±4.46 g/L,前白蛋白含量13.22±11.64 g/L,胆汁酸含量4.48±3.89 μmol/L.东北虎幼体雌雄个体间5项生化指标均无显著差异(P0.05).东北虎幼体与成体间,总蛋白、白蛋白、球蛋白含量均呈极显著差异(P<0.01).东北虎幼体与华南虎幼体间,总蛋白、球蛋白含量呈显著差异(P<0.05);白蛋白含量无显著差异(P0.05).     

东北虎幼体消化系统蛋白水解酶的初步研究

蛋白水解酶在许多生命活动中是必需的物质(Vassalli and Pepper,1994)。蛋白质的酶解修饰(Xuet al.,1999)、细胞迁移、组织再生与修复、消化系统对蛋白质的消化等均与蛋白水解酶有关(Baimbridgeet al.,1992),且蛋白水解酶功能失调会导致许多疾病(Teichertet al.,1989)。东北虎(     

东北虎血液成分的测定与分析

用氰化高铁血红蛋白法、改良纽鲍氏计算板法、显微测量法、低渗ＮａＣｌ试管法、微量毛细吸管离心法、血细胞计算板法和瑞特氏染色三区计数法，分别测得和算出８只东北虎血液中血红蛋白含量１３１±７．５ｇ／Ｌ、红细胞数７．１１±Ｏ．５３×１０１２／Ｌ、红细胞直径５．５８（４．６２—６．５５）μｍ、红细胞渗透脆性０．６０６±０．０６５—０．４３０±０．０４５％、红细胞压积容量３８．１±２．６８％、白细胞总数２３．３±７．５×１０９／Ｌ、嗜中性白细胞５７．９％、淋巴细胞３５．６％、单核细胞３．５％、嗜酸性粒细胞１．８％、嗜硷性粒细胞＜１％、平均红细胞体积５３．６±２．７５ｆＬ、平均红细胞血红蛋白含量１７．９±１．２５Ｐｇ、平均每个红细胞血红蛋白分子数１５８９±１１０×１０５个、平均红细胞血红蛋白浓度３３．５±２．７％；用全自动凯氏定氮法测定并计算得出血清总蛋白含量７３．６±１２．６ｇ／Ｌ、血清白蛋白含量４６．５±３．２％及血清球蛋白含量２７．１±１３．１ｇ／Ｌ。上述各项结果为保护东北虎提供了难得而有意义的生理参考值。     

4种生态因子对虎斑乌贼幼体生长与存活的影响

旨在研究温度、盐度、饵料种类、投饵量对虎斑乌贼（Sepia pharaonis）幼体生长与存活率的影响,以确定其生长发育的最佳生态条件,为人工育苗提供理论依据。在室内控制条件下,采用单因子试验研究了不同温度（18、21、24、27、30和33℃）、不同盐度（18、21、24、27、30和33）、不同饵料种类（虾糜、活糠虾、卤虫无节幼体、桡足类、死糠虾、虾糜＋强化卤虫后无节幼体、强化卤虫后无节幼体）、活糠虾不同投饵量（0、2、4、6、8和10 g/d）对虎斑乌贼幼体生长发育的影响。结果显示：不同温度对虎斑乌贼幼体生长影响显著（P〈0.05）,最适温度为24-27℃,在最适温度下,存活率为84.4%-91.1%,特定生长率为4.82%-6.13%,存活率（y）与温度（x）的函数关系为y=-0.15x3＋30.637x-447.002（r2=0.923）。幼体适宜盐度为24-33,最适盐度为27,在最适盐度条件下,其存活率为（90.0±5.29）%,特定生长率为（3.71±0.34）%。投喂7种开口饵料,以活糠虾效果最佳,存活率为（96.7±2.94）%,特定生长率为（3.77±0.23）%;强化卤虫后无节幼体效果次之,存活率为（95.6±2.31）%,特定生长率为（2.54±0.15）%,其余各组培养效果均不理想。投喂活糠虾,随着投饵量的增加,其存活率随之提高,摄食量（y）与个体重（x）的回归方程为y=0.227x-0.063（r2=0.921）。     

东北虎中SOX基因的检测

应用特异于HMG bOX区域的兼并引物 ,扩增了东北虎的SOX基因。在扩增产物中发现五条大小分别为 2 2 0 ,2 70 ,3 5 0 ,4 3 0和 5 60bp的扩增带。经过与地高辛标记的人SRY基因进行Southern杂交表明这五条扩增带均呈现阳性 ,说明它们均为东北虎的SOX基因片段 ,这些基因保守区的长度在基因组DNA水平上存在着明显的差异。     

东北虎,云豹,金猫红细胞上C3b受体的测定

本文通过对健康的东北虎、云豹和金猫红细胞上C_(3b)受体和免疫复合物的检测,证实这三种动物的红细胞,除了具有免疫粘附,形成免疫复合物的花环(其花环率分别为7.00、6.50和5.67)外,还可通过其膜上的C_(3b)受体结合免疫复合物,形成C_(3b)受体的花环,其花环率分别为11.50、11.88和11.83。表明东北虎、云豹和金猫的红细胞具有较强的免疫功能。     

基于目标检测的东北虎个体自动识别

东北虎个体的自动识别是种群数量评估和制定有效保护策略的重要基础。以东北虎林园和怪坡虎园38 只虎为研究对象,将目标检测方法首次应用到东北虎个体识别研究中,采用多种深度卷积神经网络模型,以实现虎个体的自动识别。首先通过相机在不同角度对 38 只东北虎进行拍摄取样,建立包含13579张图像的虎样本数据集。由于虎的体侧条纹信息不具有对称性,所以运用单次多盒目标检测（Single Shot MultiBox Detector, SSD）方法,对虎的躯干左侧条纹、右侧条纹以及脸部等不同部位图像,进行自动检测并分割提取,极大节省手工截取时间。在检测分割出的左右侧及脸部不同部位图片基础上,运用上、下、左、右平移变换进行数据增强,使图片数目扩大为原来的5 倍。采用LeNet、AlexNet、ZFNet、VGG16、ResNet34共5 种卷积神经网络模型进行个体自动识别。为了提高识别准确率,运用平均值和最大值不同组合方式来优化池化操作,并在全连接层引入概率分别为0.1、0.2、0.3、0.4的丢弃（Dropout）操作防止过拟合。实验表明,目标检测模型耗时较少,截取分割老虎不同部位条纹能达到0.6 s/张,远快于人工截取速度,并且在测试集上准确率能达到97.4%。不同姿态下的目标部位都能正确识别并分割。ResNet34模型的准确率优于其他网络模型,左右侧条纹以及脸部图像识别准确率分别为93.75%、97.01%和 86.28%,右侧条纹识别准确率优于左侧条纹和脸部图像。研究为野生虎自动相机影像的识别提供技术参考。在未来研究中,对东北虎个体影响数据进行扩充,选取更多影像数据进行训练,使网络具有更强的适应性,从而实现更准确的个体识别。     

半散养状态下东北虎交配行为的观察

在2006～2007年,采用行为取样法对泰山东北虎园7只(3雌,4雄)半散养状态下(园区占地面积20 000 m2)的东北虎的交配活动进行了为期22 d的观察,旨在了解无人干扰下东北虎的交配过程及其交配模式.结果显示:东北虎在交配过程中无锁结现象,雄虎通常在一次爬跨多次抽动后即出现射精.在1 h内出现2次射精次数的比例占总射精次数的74.0%.东北虎的交配模式属于Dewsbury分类系统中的第11种,即无锁结、有抽动、单次插入、多次射精.交配期内,每只雌虎平均日邀配28±3次,平均邀配持续时间为20.0±1.2 s;每只雄虎平均日爬跨14±1次.平均抽动持续时间为20.9±0.5 s,平均交配持续时间为45.2±1.3 s.     

中国东北虎栖息地分析与潜在生态廊道构建

东北虎（Panthera tigris altaica）是现存5个虎亚种中体型最大者,其作为全球生物多样性保护的旗舰物种,在维持健康生态系统功能中占据不可替代的重要地位。近几十年来,由于东北虎栖息地受到人类活动强烈干扰,致使栖息地破碎化,主要栖息地孤立分布,呈现岛状,天然生态廊道消失殆尽,东北虎的保护面临巨大挑战。因此,确定东北虎关键栖息地,构建与恢复东北虎栖息地之间的生态廊道十分必要。本研究运用专家模型结合东北虎栖息地选择规律和栖息地特征,综合分析植被类型、国家级与省级自然保护区分布、地形因子以及人为干扰因子共7个主要影响因子;通过层次分析法(AHP)获得各影响因子的相对权重值,运用加权线性方程获得了东北虎潜在适宜栖息地,并确定了东北虎核心分布区以及分布区间的综合代价值。通过廊道设计模型（Linkage mapper）得到东北虎核心栖息地间的潜在生态廊道。结果得到了21条东北虎潜在生态廊道,对打通国内零星分布区,特别是张广才岭-完达山-老爷岭之间的迁移通道、扩大东北虎生存空间具有现实指导意义。     

Functional and molecular mechanism of intracellular pH regulation in human inducible pluripotent stem cells

AIM To establish a functional and molecular model of the intracellular pH(pH_i) regulatory mechanism in human induced pluripotent stem cells(hiPSCs).METHODS hiP SCs(HPS0077) were kindly provided by Dr. Dai from the Tri-Service General Hospital(IRB No. B-106-09). Changes in the pH_i were detected either by microspectrofluorimetry or by a multimode reader with a pH-sensitive fluorescent probe, BCECF, and the fluorescent ratio was calibrated by the high K~+/nigericin method. NH_4Cl and Na-acetate prepulse techniques were used to induce rapid intracellular acidosis and alkalization, respectively. The buffering power(β) was calculated from the ΔpH_i induced by perfusing different concentrations of(NH_4)_2SO_4. Western blot techniques and immunocytochemistry staining were used to detect the protein expression of pH_i regulators and pluripotency markers.RESULTS In this study, our results indicated that(1) the steadystate pH_i value was found to be 7.5 ± 0.01(n = 20) and 7.68 ± 0.01(n =20) in HEPES and 5% CO_2/HCO_3~- buffered systems, respectively, which were much greater than that in normal adult cells(7.2);(2) in a CO_2/HCO_3~--buffered system, the values of total intracellular buffering power(β) can be described by the following equation: β_(tot) = 107.79(pH_i)~2-1522.2(pH_i) + 5396.9(correlation coefficient R~2 = 0.85), in the estimated pH_i range of 7.1- 8.0;(3) the Na~+/H~+ exchanger(NHE) and the Na~+/HCO_3~- cotransporter(NBC) were found to be functionally activated for acid extrusion for pHi values less than 7.5 and 7.68, respectively;(4) V-ATPase and some other unknown Na~+-independent acid extruder(s) could only be functionally detected for pHi values less than 7.1;(5) the Cl~-/OH~- exchanger(CHE) and the Cl~- /HCO_3 anion exchanger(AE) were found to be responsible for the weakening of intracellular proton loading;(6) besides the CHE and the AE, a Cl~--independent acid loading mechanism was functionally identified; and(7) in hiPSCs, a strong positive correlation was observed between the loss of pluripotency and the weakening of the intracellular acid extrusion mechanism, which included a decrease in the steady-state pH i value and diminished the functional activity and protein expression of the NHE and the NBC.CONCLUSION For the first time, we established a functional and molecular model of a pHi regulatory mechanism and demonstrated its strong positive correlation with hiPSC pluripotency.     

调控精子获能的相关因素

获能是精子发生顶体反应以及与卵子结合之前所必需的生理过程,目前精子获能的机制得到初步阐明,获能伴随着质膜重组,离子通道的调节,胆固醇的流失以及许多蛋白磷酸化状态的改变.获能同时受到内在和外在因子的调节,其中胆固醇、HCO3-、Ca2+以及蛋白磷酸化在精子获能过程中发挥着重要作用.     

Contribution of anion transporters to the acidosis-induced swelling and intracellular acidification of glial cells

This study examines the contribution of anion transporters to the swelling and intracellular acidification of glial cells from an extracellular lactacidosis, a condition well-known to accompany cerebral ischemia and traumatic brain injury. Suspended C6 glioma cells were exposed to lactacidosis in physiological or anion-depleted media, and different anion transport inhibitors were applied. Changes in cell volume and intracellular pH (pH(i)) were simultaneously quantified by flow cytometry. Extracellular lactacidosis (pH 6.2) led to an increase in cell volume to 125.1 +/- 2.5% of baseline within 60 min, whereas the pH(i) dropped from the physiological value of 7.13 +/- 0.05 to 6.32 +/- 0.03. Suspension in Cl(-)-free or HCO(3)(-)/CO(2)-free media or application of anion transport inhibitors [0.1 mM bumetanide or 0.5 mM 4, 4'-diisothio-cyanatostilbene-2,2'-disulfonic acid (DIDS)] did not affect cell volume during baseline conditions but significantly reduced cell swelling from lactacidosis. In addition, the Cl(-)-free or HCO(3)(-)/CO(2)-free media and DIDS attenuated intracellular acidosis on extracellular acidification. From these findings it is concluded that besides the known activation of the Na(+)/H(+) exchanger, activation of the Na(+)-independent Cl(-)/HCO(3)(-) exchanger and the Na(+)-K(+)-Cl(-) cotransporter contributes to acidosis-induced glial swelling and the intracellular acidification. Inhibition of these processes may be of interest for future strategies in the treatment of cytotoxic brain edema from cerebral ischemia or traumatic brain injury.     

Determination of glucose,urea and penicillin using enzyme-pH-electrodes

Conventional hydrogen ion glas electrodes have been used for the preparation of enzyme-pH-electrodes by either entrapping the enzymes within polyacrylamide gels around the electrode or as liquid layer trapped within a cellophane membrane. The enzymes were glucose oxidase, urase and penicillinase.The pH response to glucose concentration was about linear within 10^?1–10^?3 M glucose and for urea linear within 5·10^t—–5·10^?5M. The pH response to penicillin was about linear in the range from 10^?3–10^?2 M resulting in a pH shift of 1.4 units; reproduceable pH response was obtained down to concentrations of 3·10^?5 M.Studies as to the effect of buffer using an urease–pH-electrode showed a buffer concentration of 10^?2 M a substantial shift of about one pH-unit in the range of 10^?4 to 10^?2 M urea. Both urease- and penicillinase–pH-electrodes were tested as to stability showing no decrease in pH response except at high substrate concentration (1·10^?2 M) over a period of 2–3 weeks kept at room temperature.     

华南虎、东北虎、孟加拉虎的D-100p和ND5序列及其在系统进化分析中的应用

本文采用PCR和质粒克隆测序方法,获得了华南虎线粒体D-loop区的480bp序列和东北虎、孟加拉虎线粒体D-loop区的503bp序列;同时还获得了这三个虎亚种和金钱豹线粒体ND5基因5’端309bp的部分序列。根据D-loop序列分析,华南虎与孟加拉虎、东北虎的平均距离(p-distance)分别为0．11088和O．11087,而东北虎与孟加拉虎间的平均距离为0．00994;根据ND5序列分析,华南虎与孟加拉虎、东北虎的平均距离分别为0．11434和0．11758,而东北虎与孟加拉虎间的平均距离为0．00324。三个虎亚种的mtDNA D-loop和ND5序列比较表明,华南虎是这三个虎亚种中最为古老的亚种。     

The co-presence of Na+/Ca2+-K+ exchanger and Na+/Ca2+ exchanger in bovine adrenal chromaffin cells

We have previously shown that there is high Na(+)/Ca(2+) exchange (NCX) activity in bovine adrenal chromaffin cells. In this study, by monitoring the [Ca(2+)](i) change in single cells and in a population of chromaffin cells, when the reverse mode of exchanger activity has been initiated, we have shown that the NCX activity is enhanced by K(+). The K(+)-enhanced activity accounted for a significant proportion of the Na(+)-dependent Ca(2+) uptake activity in the chromaffin cells. The results support the hypothesis that both NCX and Na(+)/Ca(2+)-K(+) exchanger (NCKX) are co-present in chromaffin cells. The expression of NCKX in chromaffin cells was further confirmed using PCR and northern blotting. In addition to the plasma membrane, the exchanger activity, measured by Na(+)-dependent (45)Ca(2+) uptake, was also present in membrane isolated from the chromaffin granules enriched fraction and the mitochondria enriched fraction. The results support that both NCX and NCKX are present in bovine chromaffin cells and that the regulation of [Ca(2+)](i) is probably more efficient with the participation of NCKX.