甘氨酸对鲫鱼视网膜ERG b-波和ON型双极细胞活动的调制

本工作在分离灌流的鲫鱼视网膜上研究了甘氨酸对明,暗视视网膜电图（ＥＲＧ）ｂ－波和胞内记录的ＯＮ型双极细胞反应的作用。结果表明,甘氨酸能明显压抑ＥＲＧ ｂ－波和ＯＮ型双极细胞的反应,其作用能为士的宁所翻转;甘氨酸对用谷氨酸分离的ＥＲＧ ＰⅢ成分（光感受器电位）无明显影响。这些结果提示,甘氨酸可能直接作用于双极细胞的受体,从而调节视网膜ＯＮ通路的活动。     

NO对鲫鱼视网膜视杆和视锥信号传递的不同作用

在离体、灌流的鲫鱼视网膜上,考察了一氧化氮(NO)对视网膜电图(ERG)和水平细胞的作用.NO的供体硝普钠(SNP)压抑暗视ERGb波,却增大明视b波,这些作用均可为血红蛋白所阻遏.进而,SNP使视杆水平细胞对光反应减小,而增强L型视锥水平细胞的对光反应.这些结果表明,NO在外视网膜可压抑视杆系统的活动而增强视锥系统的活动.此外,可溶性鸟苷酸环化酶的抑制剂亚甲基蓝对视杆、视锥水平细胞的作用恰与SNP作用相反,这提示NO的作用可能由cGMP所介导.对NO的作用位点提出了工作假设.     

第二信使介导模拟低氧下丘脑CRF分泌

用模拟高原低氧的方法研究急性低氧条件下促肾上腺皮质激素释放因子（ｃｏｒｔｉｃｏｔｒｏｐｉｎ－ｒｅｌｅａｓｉｎｇｆａｃｔｏｒ,ＣＲＦ）分泌的变化及第二信使参与ＣＲＦ分泌的作用。低氧（海拔７ｋｍ）１ｈ后,正中隆起（ｍｅｄｉａｎｅｍｉｎｅｎｃｅ,ＭＥ）处ＣＲＦ含量明显下降（Ｐ＜０．０５）,下丘脑（ｈｙｐｏｔｈａｌａｍｕｓ,Ｈｙ）ＣＲＦ（不含ＭＥ）含量无明显变化,而Ｈｙ内ｃＡＭＰ含量明显增加（Ｐ＜０．０１）。脑室注射Ｆｏｒｓｋｌｉｎ、ＴＰＡ后低氧暴露（海拔５ｋｍ）１ｈ,ＭＥＣＲＦ含量下降（Ｐ＜０．０５;Ｐ＜０．０５）,ＨｙＣＲＦ无明显变化。脑室注射Ｆｏｒｓｋｌｉｎ后ＨｙｃＡＭＰ含量升高（Ｐ＜０．０５）。脑室注射Ｈ７和ＰＫＡ抑制剂,ＭＥＣＲＦ升高（Ｐ＜０．０５;Ｐ＜０．０１）,ＨｙＣＲＦ和ＨｙｃＡＭＰ均无显著变化。上述结果表明急性低氧应激中ＣＲＦ分泌显著增加,第二信使通路ＰＫＡ和ＰＫＣ通路均参与ＣＲＦ分泌     

核酶对bcl－2mRNA的体外切割

通过微机对ｂｃｌ－２ＲＮＡ二级结构的分析,设计针对ｂｃｌ－２片段５＇ＣＧＣＧＡＣＣＣＧＧＵＣＧＣＣＡＧＧＡＣＣＵＣＧ３＇的“锤头状”（Ｈａｍｍｅｒｈｅａｄ）核酶（Ｒｉｂｏｚｙｍｅ,ＲＤ）基因,平端连接于ｐＧＥＭ－３Ｚｆ（－）ＨｉｎｃⅡ位点,克隆后经测序表明序列正确,ｂｃｌ－２和Ｒｉｂｏｚｙｍｅ基因经体外转录,５０℃作用２ｈ,从１６５６－１６５７（Ｃ－Ｇ）位之间切断ｂｃｌ－２ＲＮＡ片段．     

抗真菌蛋白Rs—AFPs基因在大肠杆菌中的表达

将抗真菌蛋白Ｒｓ－ＡＦＰ１和Ｒｓ－ＡＦＰ２全长ｃＤＮＡ插入表达质粒ｐＥＴ－２２ｂ／ＮｃｏＩ＋ＳａｃＩ位点,构建成融合蛋白表达载体ｐＲＡＦ１和ｐＲＡＦ２．将不含信号肽编码序列的Ｒｓ－ＡＦＰ１和Ｒｓ－ＡＦＰ２ｃＤＮＡ分别插入ｐＥＴ－２２ｂ／Ｎｃｏｌ＋Ｓａｃｌ和ｐＥＴ－２２ｂ／Ｎｄｅｌ＋ＳａｃＩ位点,构建成不含信号肽序列的融合蛋白表达载体ｐＲＡＦ３、ｐＲＡＦ４和非融合蛋白表达载体ｐＲＡＦ５和ｐＲＡＦ６．将构建的上述各种表达载体转化Ｅ．ｃｏｌｉＢＬ２１,挑菌落培养,ＩＰＴＧ诱导,使Ｒｓ－ＡＦＰｓ基因得到表达,并用体外抑菌试验检测表达产物的活性,结果表明,各种表达载体的表达产物均具有不同程度的抑菌活性,其中,ｐＲＡＦ３和ｐＲＡＦ４表达产物的抑菌活性较明显．     

沼泽绿牛蛙的核型及银染研究

沼泽绿牛蛙的核型及银染研究樊连春,杨仲安,魏丽华,梁绍昌,桂建芳（中国科学院水生生物研究所武汉４３００７２）关键词沼泽绿牛蛙,核型,Ａｇ－ＮＯＲｓＡＮＩＮＶＥＳＴＩＧＡＴＩＯＮＯＦＴＨＥＫＡＲＹＯＴＹＰＥＡＮＤＡｇ－ＮＯＲｓＯＦＲＡＮＡＧＲＹＬＩＯ￥...     

三江平原沼泽化草甸小叶章种群地上生物量及其生长速率季节动态的…

三江平原沼泽化草甸优势种群－小叶章种群地上生物ＡＢＩ,ＡＡＧＲ,ＡＲＧＲ季节动态均呈单峰型。均至７月末到达到极大值。分别为３５８．０５ｇ／ｍ＾２,２３０．９６ｇ／ｍ＾２,１４．６８３０ｇ／ｄ,０．０６３８ｇ／ｇ．ｄ。ＢＩ,ＡＧＲ,ＲＧＲ在７月末之前为正,之后为负。     

碱性成纤维细胞生长因子对乳鼠心肌细胞缺氧复氧损伤的影响

本实验在培养的乳鼠心肌细胞缺氧复氧损伤模型上观察碱性纤维细胞生长因子对Ａ／Ｒ损伤及蛋白激酶活性的影响,以探讨ｂＥＧＦ作为药物预处理心肌保护的可行性及其机理。结果表明,ｂＥＧＦ预处理呈浓度依赖地提高Ａ／Ｒ后心肌细胞存活率,减少细胞内ＡＴＰ消耗及胞浆乳酸脱氢酶漏出;ＰＫＣ抑制剂Ｈ７完全消除ｇＦＧＦ的上述保护作用;实验结果表明,ｂＦＧＦ可以直接激活心肌细胞ＰＫＣ,其激活时相的变化与缺氧预处理者相近,提示     

苦瓜籽核糖体失活蛋白的理化性质及生物活性

采用硫酸铵分级分离,假配基亲和层析和ＳｅｐｈａｃｒｙｌＳ－１００分子筛层析等方法,从苦瓜籽中获得核糖体失活蛋白（ＲＩＰ）．经ＳＤＳ－ＰＡＧＥ、ＰＡＧＥ、ＩＥＦ和ＰＡＳ方法分析均表明为单一蛋白着色带或单一糖蛋白着色带．根据ＳＤＳ－ＰＡＧＥ和Ｓｅｐｈａｄｅｘ Ｇ－１５０分子筛层析结果计算其相对分子质量为３．０×１０４,经ＩＥＦ－ＰＡＧＥ结果计算其ｐＩ为８．９～９．０．对无细胞系统中蛋白质生物合成抑制活性明显,其ＩＣ５０为５．３×１０－ １０ ｍ ｏｌ／Ｌ左右．体外生物活性试验结果表明其对人肝癌细胞、Ｖｅｒｏ、ＳＰ２／０、３Ｔ３、Ｋｂ、Ｎａｖａｎａ 等肿瘤细胞株均表现有不同程度的抑制作用．而对完整细胞人胚肺二倍体细胞却毒性极小．因此,上述实验结果为该ＲＩＰ的进一步深入研究和有可能开发成免疫毒素的高效弹头药物提供了一定的工作基础．     

低钙对鲫鱼视网膜中视杆,视锥信号向水平细胞传递的不同作用

本工作应用细胞内记录技术,在灌流的鲫鱼离体视网膜的标本上,研究了低钙对外网状层中视锥、视杆信号向水平细胞传递的不同影响。当降低灌流溶液中Ｃａ（２＋）浓度至０．１ｍｍｏｌ／Ｌ时使视锥－和视杆－水平细胞（Ｃｏｎｅ－ＨＣ和Ｒｏｄ－ＨＣ）均去极化,但对两者的光反应性有不同的影响：使Ｌ型Ｃｏｎｅ－ＨＣ对６８０ｕｍ和５００ｕｍ闪光反应的幅度有不同程度的减小,但使Ｒｏｄ－ＨＣ对光反应的幅度显著增大。在０．１ｍｍｏｌ／ＬＣａ（２＋）Ｒｉｎｇｅｒ液灌流时,无论是明视ＰⅢ（反映机锥活动）或暗视ＰⅢ（反映视杆活动）,其反应的幅度均增大,提示发生在低钙时Ｃｏｎｅ－ＨＣ和Ｒｏｄ－ＨＣ光反应性的不同变化产生于突触传递的过程中。进而,磷酸二酯酶抑制剂ＩＢＭＸ对ＰⅢ的影响与０．１ｍｍｏｌ／ＬＣａ（２＋）Ｒｉｎｇｅｒ液的作用相似,而对Ｌ型Ｃｏｎｅ－ＨＣ和Ｒｏｄ－ＨＣ的作用均是使光反应性增大。这提示,在０．１ｍｍｏｌ／ＬＣａ（２＋）的Ｒｉｎｇｅｒ液中,Ｃｏｎｅ－ＨＣ和Ｒｏｄ－ＨＣ光反应性的变化差异可能是由于低钙对视锥和视杆终末Ｃａ２＋内流的不同影响而产生。     

Post-irradiation hypoxic incubation of X-irradiated MOLT-4 cells reduces apoptotic cell death by changing the intracellular redox state and modulating SAPK/JNK pathways

To elucidate radiobiological effects of hypoxia on X-ray-induced apoptosis, MOLT-4 cells were treated under four set of conditions: (1) both X irradiation and incubation under normoxia, (2) X irradiation under hypoxia and subsequent incubation under normoxia, (3) X irradiation under normoxia and subsequent incubation under hypoxia, and (4) both X irradiation and incubation under hypoxia, and the induction of apoptosis was examined by fluorescence microscopy. About 28–33% apoptosis was observed in cells treated under conditions 1 and 2, but this value was significantly reduced to around 18–20% in cells treated under conditions 3 and 4, suggesting that post-irradiation hypoxic incubation rather than hypoxic irradiation mainly caused the reduction of apoptosis. The activation and expression of apoptosis signal-related molecules SAPK/JNK, Fas and caspase-3 were also suppressed by hypoxic incubation. Effects of hypoxic incubation were canceled when cells were treated under conditions 3 and 4 with an oxygen-mimicking hypoxic cell radiosensitizer, whereas the addition of N-acetyl-L-cysteine again reduced the induction of apoptosis. From these results it was concluded that hypoxia reduced the induction of apoptosis by changing the intracellular redox state, followed by the regulation of apoptotic signals in X-irradiated MOLT-4 cells.     

Electrophysiological consequences of leukotrienes applied on isolated rat retina

Scotopic vision is the result of a cascade of light-dependent biochemical events in rod outer segments (ROS) involving mainly a cGMP-modulation of sodium current. This modification of ionic currents induces changes of membrane potential which generates electroretinographic (ERG) waves. As (i) ERG disturbances are commonly recorded in hypoxic and inflammatory retinal diseases (ii) leukotrienes (LTs), a very potent mediators of inflammation, disturb ionic exchanges in several artificial or natural membrane systems, we undertook the investigation of the effects of LTs on ERG record in mammalian isolated retina. LTB4, LTC4 and LTD4, all induced a dose-dependent marked reduction of the b wave amplitude of ERG. This effect is correlated with a significant decrease in the survival time of the retina. The analysis of the modification of ERG indicates that LTs exhibit a real toxic effect since b wave is mainly affected while P III wave is unchanged. Comparatively with other nervous cells, this phenomenon may be attributed to an increase in Na+ permeability of ROS. It is suggested that LTs may be involved in the development of inflammatory or ischemic retinal diseases.     

Activator of G Protein Signaling 8 (AGS8) Is Required for Hypoxia-induced Apoptosis of Cardiomyocytes: ROLE OF G�¦� AND CONNEXIN 43 (CX43)*

Ischemic injury of the heart is associated with activation of multiple signal transduction systems including the heterotrimeric G-protein system. Here, we report a role of the ischemia-inducible regulator of Gβγ subunit, AGS8, in survival of cardiomyocytes under hypoxia. Cultured rat neonatal cardiomyocytes (NCM) were exposed to hypoxia or hypoxia/reoxygenation following transfection of AGS8siRNA or pcDNA::AGS8. Hypoxia-induced apoptosis of NCM was completely blocked by AGS8siRNA, whereas overexpression of AGS8 increased apoptosis. AGS8 formed complexes with G-proteins and channel protein connexin 43 (CX43), which regulates the permeability of small molecules under hypoxic stress. AGS8 initiated CX43 phosphorylation in a Gβγ-dependent manner by providing a scaffold composed of Gβγ and CX43. AGS8siRNA blocked internalization of CX43 following exposure of NCM to repetitive hypoxia; however it did not influence epidermal growth factor-mediated internalization of CX43. The decreased dye flux through CX43 that occurred with hypoxic stress was also prevented by AGS8siRNA. Interestingly, the Gβγ inhibitor Gallein mimicked the effect of AGS8 knockdown on both the CX43 internalization and the changes in cell permeability elicited by hypoxic stress. These data indicate that AGS8 is required for hypoxia-induced apoptosis of NCM, and that AGS8-Gβγ signal input increased the sensitivity of cells to hypoxic stress by influencing CX43 regulation and associated cell permeability. Under hypoxic stress, this unrecognized response program plays a critical role in the fate of NCM.     

AHR,a novel acute hypoxia‐response sequence,drives reporter gene expression under hypoxia in vitro and in vivo

ADAMTS1 (a disintegrin and metalloproteinase with thrombospondin motifs 1) is an early immediate gene. We have previously reported that ADAMTS1 was strongly induced by hypoxia. In this study, we investigated whether ADAMTS1 promoter‐driven reporter signal is detectable by acute hypoxia. We constructed the GFP (green fluorescent protein) expression vector [AHR (acute hypoxia‐response sequence)‐GFP] under the control of ADAMTS1 promoter and compared it with the constitutive GFP‐expressing vector under the control of CMV (cytomegalovirus promoter‐GFP). We transduced AHR‐GFP and examined whether GFP signals can be detected under the acute hypoxia. When the human umbilical vein [HUVEC (human umbilical vein endothelial cells)] was transduced under normoxia, there were few GFP signals, while CMV‐GFP showed considerable GFP signals. When HUVEC was stimulated with hypoxia, GFP signals from AHR‐GFP gene were induced under hypoxic conditions. Notably, the GFP signals peaked at 3 h under hypoxia. In ischaemic hind limb model, transduced AHR‐GFP showed hypoxic induction of GFP signals. In summary, we have demonstrated that the AHR system induced the reporter gene expression by acute hypoxia, and its induction is transient. This is the first report showing the unique acute hypoxia‐activated gene expression system.     

Human Retinal Light Sensitivity and Melatonin Rhythms Following Four Days in Near Darkness

The rods in the retina are responsible for night vision, whereas the cone system enables day vision. We studied whether rod function in humans exhibits an endogenous circadian rhythm and if changes occur in conditions of prolonged darkness. Seven healthy subjects (mean age±SD: 25.6±12.3 yr) completed a 4.5‐day protocol during which they were kept in complete darkness (days 1 and 4) and near darkness (<0.1 lux red light, days 2 and 3). Electroretinography (ERG) and saliva collections were done at intervals of at least 3 h for 27 h on days 1 and 4. Full‐field ERGs were recorded over 10 low‐intensity green light flashes known to test predominantly rod function. As a circadian marker, salivary melatonin concentration was measured by radioimmunoassay. The ERG data showed that rod responsiveness to light progressively diminished in darkness (significantly lower a‐ and b‐wave amplitudes, longer b‐wave implicit time). The decrease in amplitude (b‐wave) from day 1 to day 4 averaged 22±14%. After correction for the darkness‐related linear trend, the circadian variations in ERG indices were weak and usually non‐significant, with slightly higher responsiveness to light during the day than night. Rod sensitivity (by K index) tended to decrease. Strikingly, the overall amount of melatonin secretion (area under 24 h curve) also decreased from day 1 to day 4 by 33.1±18.9% (p=.017). The drift of the melatonin rhythm phase was within the normal range, less than 56 min over three days. There was no significant correlation between the changes in ERG responses and melatonin. In conclusion, scotopic retinal response to (low‐intensity) light and the amount of melatonin secreted are diminished when humans are kept in continuous darkness. Both processes may have a common underlying mechanism implicating a variety of neurochemicals known to be involved in the regulation of both photoreceptor and pineal gland function.     

Electrophysiological consequences of leukotrienes applied on isolated rat retina

Scotopic vision is the result of a cascade of light-dependent biochemical events in rod outer segments (ROS) involving mainly a cGMP-modulation of sodium current. This modification of ionic currents induces changes of membrane potential which generates electroretinographic (ERG) waves. As (i) ERG disturbances are commonly recorded in hypoxic and inflammatory retinal diseases (ii) leukotrienes (LTs), a very potent mediators of inflammation, disturb ionic exchanges several artificial or natural membrane systems, we undertook the investigation of the effects of LTs on ERG record in mammalian isolated retina. LTB₄, LTC₄ and LTD₄, all induced a dose-dependent marked reduction of the b wave amplitude of ERG. This effect is correlated with a significant decrease in the survival time of the retina. The analysis of the modification of ERG indicates that LTs exhibit a real toxic effect since b wave is mainly affected while P III wave is unchanged. Comparatively with other nervous cells, this phenomenon may be attributed to an increase in Na⁺ permeability of ROS. It is suggested that LTs may be involved in the development of inflammatory or ischemic retinal diseases.     

Adaptive effects of hypoxic preconditioning in brain neurons

A preventive short-term hypoxia (preconditioning) increases neuronal resistance against subsequent strong hypoxic effects. Literature review and authors' own data on molecular-cellular mechanisms of the hypoxic preconditioning, are presented. Participation of intracellular signal transduction, genome, stress-proteins, and neuromodulating peptides in this process, is discussed. The role of glutamatergic as well as calcium and phosphoinositide regulatory systems and neuromodulating factors as the components of a "volume" signal transmission are analyzed in hypoxic precondition-associated induction of functional tolerance mechanisms against acute harmful effects in neurones of olfactory slices.     

The in vitro sensitivity of chronically hypoxic EMT6/SF cells to X-radiation and hypoxic cell radiosensitizers

We have investigated the effect of extreme, prolonged hypoxia on the radiosensitivity of EMT6/SF cells in vitro. As cells were kept hypoxic for 1-24 h, their radiosensitivity increased, but no further change was noted for hypoxic incubation beyond 24 h. Chronically hypoxic (45 h) cells were more radiosensitive than acutely hypoxic (1 h) cells by a factor of 1.43. When chronically hypoxic cells were re-aerated, the increased radiosensitivity persisted, although it was reduced. Misonidazole (MISO) radiosensitization was equally effective under conditions of acute and chronic hypoxia. In contrast, MISO, SR2555 and SR2508 were more cytotoxic in chronically hypoxic cultures than in acutely hypoxic cells. Measurements suggested that intracellular thiols may play an important role in the effects observed.     

Hypoxia reveals posterior thalamic, cerebellar, midbrain, and limbic deficits in congenital central hypoventilation syndrome.

Congenital central hypoventilation syndrome (CCHS) patients show deficient respiratory and cardiac responses to hypoxia and hypercapnia, despite apparently intact arousal responses to hypercapnia and adequate respiratory motor mechanisms, thus providing a model to evaluate functioning of particular brain mechanisms underlying breathing. We used functional magnetic resonance imaging to assess blood oxygen level-dependent signals, corrected for global signal changes, and evaluated them with cluster and volume-of-interest procedures, during a baseline and 2-min hypoxic (15% O(2), 85% N(2)) challenge in 14 CCHS and 14 age- and gender-matched control subjects. Hypoxia elicited significant (P < 0.05) differences in magnitude and timing of responses between groups in cerebellar cortex and deep nuclei, posterior thalamic structures, limbic areas (including the insula, amygdala, ventral anterior thalamus, and right hippocampus), dorsal and ventral midbrain, caudate, claustrum, and putamen. Deficient responses to hypoxia included no, or late, changes in CCHS patients with declining signals in control subjects, a falling signal in CCHS patients with no change in controls, or absent early transient responses in CCHS. Hypoxia resulted in signal declines but no group differences in hypothalamic and dorsal medullary areas, the latter being a target for PHOX2B, mutations of which occur in the syndrome. The findings extend previously identified posterior thalamic, midbrain, and cerebellar roles for normal mediation of hypoxia found in animal fetal and adult preparations and suggest significant participation of limbic structures in responding to hypoxic challenges, which likely include cardiovascular and air-hunger components. Failing structures in CCHS include areas additional to those associated with PHOX2B expression and chemoreceptor sites.