大鼠不同脑区突触体钙水平的年龄差异

本实验使用荧光指示剂Ｆｕｒａ－２与Ｔｂ~（３＋）,检测了不同年龄组大鼠的不同脑区（海马、皮层、间脑、小脑）突触体内游离钙与膜结合钙水平。结果显示,与青年对照组相比,老年大鼠大部分脑区（海马、皮层、间脑）突触体内游离钙水平显著增高,尤其是海马突触体内游离钙增高极为显著;其突触体膜结合钙水平表现为：海马、小脑两脑区明显升高,而皮层、间脑两脑区明显下降,呈现一种全脑范围内的钙水平失衡。提示动物的衰老与其脑内钙自体平衡失调有关。     

The 3-D structure of a zinc metallo-beta-lactamase from Bacillus cereus reveals a new type of protein fold.

The 3-D structure of Bacillus cereus (569/H/9) beta-lactamase (EC 3.5.2.6), which catalyses the hydrolysis of nearly all beta-lactams, has been solved at 2.5 A resolution by the multiple isomorphous replacement method, with density modification and phase combination, from crystals of the native protein and of a specially designed mutant (T97C). The current model includes 212 of the 227 amino acid residues, the zinc ion and 10 water molecules. The protein is folded into a beta beta sandwich with helices on each external face. To our knowledge, this fold has never been observed. An approximate internal molecular symmetry is found, with a 2-fold axis passing roughly through the zinc ion and suggesting a possible gene duplication. The active site is located at one edge of the beta beta sandwich and near the N-terminal end of a helix. The zinc ion is coordinated by three histidine residues (86, 88 and 149) and a water molecule. A sequence comparison of the relevant metallo-beta-lactamases, based on this protein structure, highlights a few well-conserved amino acid residues. The structure shows that most of these residues are in the active site. Among these, aspartic acid 90 and histidine 210 participate in a proposed catalytic mechanism for beta-lactam hydrolysis.     

正常人各年龄组染色体着丝粒点(Cd)研究

本文运用本室改良的Cd-NOR银染技术对80例4个年龄组的正常中国人的Cd变化进行了较系统的研究, 结果表明:(1)正常人随年龄增加,Cd消失的频率、Cd变异及Cd-NOR融合频率也相应增加,特别是Ⅲ、Ⅳ组(中、老年组)增加的频率尤为显著;(2)首次对Cd消失的过程提出了独特的观点,即Cd消失首先表现为Cd变小, 随着变小程度的加大,最终导致Cd消失;(3)在本研究中首次观察到单个Cd的现象,作者认为是细胞分裂中期染色体着丝一分为二的延迟现象。各年龄组间单Cd出现频率无统计学差异,同一年龄组中,2号染色体和1号染色体上单Cd出现频率显著高于理论值;(4)随年龄增高,Cd各项观察值的增高在男性与女性间未见明显的差异。 Abstract:The Cd variation of human chromosome in four groups of different age has been investigated.The result shows that the frequencies of Cd disappearing,size variation and Cd-NOR fusion increased with the age rising,especially in the group of aged people.We suggest that the variation of Cd shows the size changes first,and then disappears completely.We also observed some cells in which a few chromosomes shows only a single Cd in centromeric region.Cd variation in different age groups has no significant difference between the male and the female.     

A role for cAMP in angiotensin II mediated inhibition of cell growth in AT1A receptor-transfected CHO-K1 cells

G-protein coupled Angiotensin II receptors (AT_1A), mediate cellular responses through multiple signal transduction pathways. In AT_1A receptor-transfected CHO-K1 cells (T3CHO/AT_1A), angiotensin II (AII) stimulated a dose-dependent (EC₅₀=3.3 nM) increase in cAMP accumulation, which was inhibited by the selective AT₁, nonpeptide receptor antagonist EXP3174. Activation of protein kinase C, or increasing intracellular Ca²⁺ with ATP, the calcium ionophore A23187 or ionomycin failed to stimulate cAMP accumulation. Thus, AII-induced cAMP accumulation was not secondary to activation of a protein kinase C- or Ca²⁺/calmodulin-dependent pathway. Since cAMP has an established role in cellular growth responses, we investigated the effect of the AII-mediated increase in cAMP on cell number and [³H]thymidine incorporation in T3CHOA/AT_1A cells. AII (1 M) significantly inhibited cell number (51% at 96 h) and [³H]thymidine incorporation (68% at 24 h) compared to vehicle controls. These effects were blocked by EXP3174, confirming that these responses were mediated through the AT₁ receptor. Forskolin (10 M) and the cAMP analog dibutyryl-cAMP (1 mM) also inhibited [³H]thymidine incorporation by 55 and 25% respectively. We extended our investigation on the effect of AII-stimulated increases in cAMP, to determine the role for established growth related signaling events, i.e., mitogen-activated protein kinase activity and tyrosine phosphorylation of cellular proteins. AII-stimulated mitogen-activated protein kinase activity and phosphorylation of the 42 and 44 kD forms. These events were unaffected by forskolin stimulated increases in cAMP, thus the AII-stimulated mitogen-activated protein kinase activity was independent of cAMP in these cells. AII also stimulated tyrosine phosphorylation of a number of cellular proteins in T3CHO/AT_1A cells, in particular a 127 kD protein. The phosphorylation of the 127 kD protein was transient, reaching a maximum at 1 min, and returning to basal levels within 10 min. The dephosphorylation of this protein was blocked by a selective inhibitor of cAMP dependent protein kinase A, H89-dihydrochloride and preexposure to forskolin prevented the AII-induced transient tyrosine phosphorylation of the 127 kD protein. These data suggest that cAMP, and therefore protein kinase A can contribute to AII-mediated growth inhibition by stimulating the dephosphorylation of substrates that are tyrosine phosphorylated in response to AII.     

A role for natural simian immunodeficiency virus and human immunodeficiency virus type 1 nef alleles in lymphocyte activation.

A T-lymphoid cell line termed 221 was derived from a rhesus monkey infected with herpesvirus saimiri. Growth of 221 cells was dependent on the addition of interleukin-2 (IL-2) to the culture medium. In the absence of IL-2, 221 cells arrested in G0-G1 but did not die. Simian immunodeficiency virus (SIV) replicated efficiently in IL-2-stimulated 221 cells whether or not the nef gene was present. In the absence of IL-2, nef-containing SIV replicated 8 to 100 times more efficiently in 221 cells than did the same virus lacking nef. nef-containing virus preferentially stimulated the production of IL-2 from 221 cells. HIV-1 nef and v-ras genes, but not the c-ras gene, were shown to substitute functionally for SIV nef when tested as recombinant viruses in this assay system. These results demonstrate a role for natural nef in causing lymphoid cell activation, and they provide a system for delineating the biochemical mechanisms responsible for this activation.     

Potent inhibition of human immunodeficiency virus type 1 in primary T cells and alveolar macrophages by a combination anti-Rev strategy delivered in an adeno-associated virus vector.

The rate of viral replication appears to play a pivotal role in human immunodeficiency virus type 1 (HIV-1) pathogenesis and disease progression as it outstrips the capacity of the immune system to respond. Important cellular sites for HIV-1 production include T lymphocytes and tissue macrophages. Antiviral strategies, including newer treatment modalities such as gene therapy of HIV-1-susceptible cell populations, must be capable of engendering durable inhibitory effects to HIV-1 replication in both of these primary cell types in order to be effective. Among the potential genetic targets for intervention in the HIV-1 life cycle, the Rev regulatory system, consisting of Rev and its binding site, the Rev-responsive element (RRE), stands out as particularly attractive. Rev is essential for maintaining the stability of the viral genomic RNA as well as viral mRNAs encoding key structural and regulatory proteins. Moreover, it exhibits favorable threshold kinetics, in that Rev concentrations must rise above a critical level to exert their effect. To disable Rev function, primary T cells or macrophages were transduced with anti-Rev single-chain immunoglobulin (SFv) or RRE decoy genes either singly or in combination by employing adeno-associated virus vectors and then challenged with HIV-1. By directing both a protein and a nucleic acid against the normal interaction between Rev and the RRE, this genetic antiviral strategy effectively inhibited infection by either clinical or laboratory virus isolates. These results provide a framework for novel interventions to reduce virus production in the infected host.     

Two distinct CCR5 domains can mediate coreceptor usage by human immunodeficiency virus type 1.

The chemokine receptor CCR5 is the major fusion coreceptor for macrophage-tropic strains of human immunodeficiency virus type 1 (HIV-1). To define the structures of CCR5 that can support envelope (Env)-mediated membrane fusion, we analyzed the activity of homologs, chimeras, and mutants of human CCR5 in a sensitive gene reporter cell-cell fusion assay. Simian, but not murine, homologs of CCR5 were fully active as HIV-1 fusion coreceptors. Chimeras between CCR5 and divergent chemokine receptors demonstrated the existence of two distinct regions of CCR5 that could be utilized for Env-mediated fusion, the amino-terminal domain and the extracellular loops. Dual-tropic Env proteins were particularly sensitive to alterations in the CCR5 amino-terminal domain, suggesting that this domain may play a pivotal role in the evolution of coreceptor usage in vivo. We identified individual residues in both functional regions, Asp-11, Lys-197, and Asp-276, that contribute to coreceptor function. Deletion of a highly conserved cytoplasmic motif rendered CCR5 incapable of signaling but did not abrogate its ability to function as a coreceptor, implying the independence of fusion and G-protein-mediated chemokine receptor signaling. Finally, we developed a novel monoclonal antibody to CCR5 to assist in future studies of CCR5 expression.     

Effects of acute hyperoxic exposure on solute fluxes across the blood-gas barrier in rat lungs

Zheng, Lu P., Rui Sheng Du, and Barbara E. Goodman.Effects of acute hyperoxic exposure on solute fluxes across the blood-gas barrier in rat lungs. J. Appl.Physiol. 82(1): 240-247, 1997.We investigatedeffects of acute hyperoxia on solute transport from air space tovascular space in isolated rat lungs. Air spaces were filled withKrebs-Ringer bicarbonate solution containing fluoresceinisothiocyanate-labeled dextran (FD-20; mol wt 20,000) and either²²Na⁺and [¹⁴C]sucrose, orD-[¹⁴C]glucoseandL-[³H]glucose.Apparent permeability-surface area products for tracers over time (upto 120 min) were calculated for isolated perfused lungs from controlrats (room air) and rats exposed to >95%O₂ for 48 or 60 h immediatelypostexposure. After O₂ exposures,mean fluxes for[¹⁴C]sucrose and FD-20were significantly higher than in room-air control lungs. However,amiloride-sensitive Na⁺ and activeD-glucose fluxes were unchangedafter hyperoxic exposure. Therefore, it is unlikely that decreases innet solute transport in this lung-injury model contributed to pulmonaryedema resulting from O₂ toxicity.Increased net solute transport shown to help resolve pulmonary edemaafter acute hyperoxic exposure must therefore begin during the recoveryperiod. In summary, our data show increases in passive solute fluxesbut no changes in active solute fluxes immediately after acutehyperoxic lung injury.