人细胞差异表达基因cDNA消减杂交体系的建立

为克隆肺腺癌分化相关基因, 采用诱导分化与消减杂交相结合的策略, 建立了全反式维甲酸(RA)诱导前后人肺腺癌细胞系的cDNA消减文库, 得到124个cDNA消减克隆. 经加减法杂交差异筛选、DNA和RNA印迹、cDNA全序列测定和生物学功能分析, 分离到3个在人肺腺癌细胞系分化过程中由RA激活而特异表达的新的cDNA序列这一策略和技术路线适用于分离细胞中呈过量表达或表达抑制基因的cDNA克隆, 并具有反映细胞分化过程中基因表达动态变化特征和相对简便适用的特点.     

A comparative study of the effects of iloprost and PGE1 on pulmonary arterial pressure and edema formation in the isolated perfused rat lung model

Isolated lungs from male Wistar rats (250–350 g) were perfused at a constant flow rate (10 ml/min, non -recirculating) with Krebs-Ringerbicarbonate buffer containing 4.5 % bovine serum albumin, and were ventilated at a positive pressure (60 breaths/min). Pulmonary arterial pressure and lung weight (as a measure of edema formation) were recorded continuously. After an equilibration period of 20 minutes the various test compounds were added to the perfusion fluid and experimental recording was continued for another 60 minutes.The effects of the stable PGI₂-mimetic, iloprost, of PGE₁, and of the biologically active PGE₁-metabolite, 13,14-dihydro-PGE,, were evaluated in this model (n=6). Iloprost showed slight, but not significant vasodilation; however, lung weight remained unchanged. PGE₁ and 13,14-dihydro-PGE₁ also caused slight vasodilation, but in contrast to iloprost these compounds induced distinct pulmonary edema. The lung weight gain was discernible at concentrations of 2.8 × 10^-6 mol/1 (significant at 2.8 × 10^-5 mol/l; p 0.05) and was accompanied by increases in the wet-weight to dry-weight ratios. These findings were duplicated in a second set of experiments (n = 6) from which the same results were obtained.The results indicate that at high concentrations PGE, (and 13,14-dihydro-PGE₁), but not iloprost, can induce pulmonary edema in rats probably by increasing the permeability of the pulmonary vasculature.     

The Nucleotide Sequence of Human Acylamino Acid-Releasing Enzyme

The nucleotide sequence of a cDNA coding for the human acylaminoacid-releasing enzyme (AARE, also known as acylpeptide hydrolase)[EC 3.4.19.1] subunit has been determined. The amino acid sequenceof human AARE subunit deduced from its cDNA nucleotide sequenceshowed a high degree of identity (91.5%) with both the correspondingproteins from the pig and the rat. The AARE cDNA shows 99.2%identity with a 3.3 kb cDNA transcribed from a locus (DNF15S2)on the short arm of human chromosome 3, whose deletion is associatedwith small cell lung cancer, taking into consideration thatthe sequence of the 3.3-kb cDNA previously reported was causedby misreading.     

Detection of oligoclonal T lymphocytes in lymph nodes draining from advanced non-small-cell lung cancer

Despite the combined use of surgery and chemoradiotherapy, the poor prognosis of advanced non-smallcell lung cancer (NSCLC) requires the definition of new therapeutic approaches. The presence of T lymphocytes, with peculiar phenotypic, functional and molecular characteristics within the tumour, suggested the possible use of these cells, expanded in vitro, in protocols of adoptive immunotherapy. We have described how a population of oligoclonal T lymphocytes, derived from advanced NSCLC, can be expanded in vitro and has the capability of lysing autologous cancer cells. What is more important, we observed that patients with advanced NSCLC, treated with TIL expanded in vitro and recombinant interleukin-2, seemed to have a disease-free period longer than that of patients treated with conventional chemoradiotherapy. in an attempt to find new sources of specific lymphocytes for immunotherapy, we describe the analysis of the phenotypic, functional and molecular characteristics of T lymphocytes, derived from lymph nodes draining advanced NSCLC. In this paper we show that these cells, have restriction patterns of T cell receptor chain similar to those detectable in the population of infiltrating T lymphocytes. This finding suggests that T cells derived from draining lymph nodes of advanced NSCLC have peculiar characteristics and can be a suitable source of effector cells for protocols of adoptive immunotherapy in lung cancer treatment.     

大鼠胸部照射后支气管肺泡灌洗液成分和肺组织纤溶活力的变化

大鼠胸部照射γ射线２０Ｇｙ,照射后观察支气管肺泡灌洗液（ＢＡＬＦ）中蛋白质含量、细胞总数和分类及巨噬细胞存活率,肺指数及肺组织纤溶活力,并做了大体解剖和组织学检查。结果为：照射后２周开始ＢＡＬＦ中蛋白质含量、细胞总数和中性粒细胞即明显增多,照射后１——２个月持续处于高水平,高峰在１．５─２个月,照射后３─４个月有所下降,肺指数有相应变化;肺组织纤溶活力则相反,从照射后２周开始持续下降,至照射后２个月已降至最低,接近于零。形态学观察也见到有早期肺水肿和晚期肺萎缩等变化。由上结果可见,大鼠胸部照射后的早期主要为渗出性病变和纤溶活力的降低。文中讨论了照射后血管内皮细胞的损伤在放射性肺损伤发病中的作用。     

Growth characteristics,morphology, and phospholipid composition of human type II pulmonary alveolar cells grown in a collagen-free microenvironment

Summary Human lung epithelial cells have been isolated and maintained in pure culture and characterized during their time in culture. Any residual fibroblasts were removed by selective trypsinization within the first 48 h in culture and the residual epithelial cells from the primary culture grew to confluent density. The epithelial cells at Passage 2 or greater were serially subpassaged when cultures reached ca. 80% confluency. This procedure permitted us to conduct biochemical and structural studies of starting materials and subsequent population doublings. Electron microscope evaluation of both initial monolayers and cell suspensions showed cultures to be composed of a single cell type. These cells had microvilli on their free or apical surface. Subsequent population doubling level 1 up to 5 exhibited the same structures. They contained lamellar inclusions, which are typical of Type II alveolar epithelial cells. Fetal lung (age 18 to 20 wk) cell suspensions processed for electron microscopy before culturing showed cells to be undifferentiated, epithelial-like with small microvilli along cell borders, and with desmosomes at cell junctions. Lamellar inclusions were not observed in these cells. Ultrastructural studies of the cultured epithelial cells demonstrated that the lamellar inclusions had a slightly positive reaction when tested for acid phosphatase. Phospholipid analysis of these lung epithelial cells showed a phospholipid composition consistent with that found in surfactant-containing Type II cells. Cultured epithelial cells stained with phosphine 3-R demonstrated a green fluorescent cytoplasm and nucleus with brightly fluorescent yellow-orange perinuclear particles. The preceding characterization of these cells leads us to conclude that they exhibit structural and biochemical features commensurate with Type II epithelial cells from human lung. Moreover, these selection techniques applied to the isolation of human lung Type II cells from the tissue permit us to study the differentiative function of these cells routinely under conditions of growth in vitro. This work was supported in part by grants from EPA, R 806638-01 and 131-640-1599A1     

Effect of lipids on activity and conformation of lysolecithin:lysolecithin acyltransferase from rabbit lung

Summary Lysolecithin:lysolecithin acyltranferase is an enzyme which in several previous studies has shown a dual behavior catalyzing two types of reaction, transacylation or hydrolysis, with the same substrate. Both activities have shown to be dependent on several environmental conditions and among them, the presence of lipids.The addition of several classes of lipids activated in all the cases the enzyme, decreasing the hydrolysis/transacylation molar ratio. This effect was higher for PC/PE/Chol mixture than for other lipids assayed. Circular dichroism spectra of the enzyme did not show any change with the addition of lipids, concluding that the effect of lipids was not due to any structural change in the protein. The hypothesis has been made of an influence of lipids on the physical state of the substrate as well as, possibly, on the enzyme-substrate interaction.The significance of these effects on the physiological role of lysolecithin:lysolecithin acyltransferase from soluble fraction of rabbit lung is discussed.Abbreviations Chol cholesterol - CMC critical micellar concentration - DPPC dipalmitoylphosphatidylcholine - FA fatty acid - H/T hydrolysis/transacylation molar ratio - LPC lysophosphatidylcholine - PC phosphatidylcholine - PE phosphatidylethanolamine - TG triglyceride - UV ultraviolet     

Endogenous proteinases modulate the function of the β-adrenergic receptor-adenylate cyclase system

Photoaffinity labeling techniques have recently demonstrated that mammalian β₁- and β₂-adrenergic receptors reside on peptides of M_r 62 000–64 000. These receptor peptides are susceptible to endogenous metalloproteinases which produce peptides of M_r 30 000–55 000. Several proteinase inhibitors markedly attenuate this process, specifically EDTA and EGTA. In this study we investigated the functional significance of this proteolysis (and its inhibition) in the β₂-adrenergic receptor-adenylate cyclase system derived from rat lung membranes. Membrane preparations containing proteolytically derived fragments of the receptor of M_r 40000–55 000 are fully functional with respect to their ability to bind β-adrenergic antagonist radioligands such as [³H]dihydroalprenolol and β-adrenergic antagonist photoaffinity reagents such as p-azido-m-[^{125I]iodobenzylcarazolol}. They retain the ability to form a high-affinity, agonist-promoted, guanine nucleotide-sensitive complex thought to represent a ternary complex of agonist, receptor and guanine nucleotide regulatory protein. Nonetheless, after proteolysis, GTP is less able to revert this high-affinity receptor complex to one of lower affinity, and all aspects of adenylate cyclase stimulation are reduced. In addition, the functional integrity of the N protein in membranes prepared without proteinase inhibitors is reduced as assessed by reconstitution studies with the cyc[su− variant of S49 lymphoma cell membranes. These results suggest that endogenous proteolysis does not directly impair the ability of β-adrenergic receptors to either bind ligands or interact with the guanine nucleotide regulatory protein. However, they imply that endogenous proteolysis likely impairs the functionality of other components of the adenylate cyclase system, such as the nucleotide regulatory protein.     

Responses of the teleost Hoplias malabaricus to hypoxia

Synopsis Oxygen uptake (VO₂) during graded hypoxia, rate of hypoxia acclimation, breathing frequency (f_R), breath volume (V_{S, R}) and gill ventilation (V_G) were measured in Hoplias malabaricus. Normoxia and hypoxia acclimated fish had similar and constant VO₂ and V_G in a range of water PO₂ from 150 to 25 mmHg. Hypoxia acclimated fish showed significantly higher VO₂ in severe hypoxia (PO₂ <15 mmHg). Normoxia acclimated fish showed symptoms similar to hypoxic coma after 1 h of exposure to water PO₂ of 10 mmHg whereas the same symptoms were observed only at PO₂ of 5 mmHg for fish acclimated to hypoxia. Fish required 14 days to achieve full acclimation to hypoxia (PO₂ ≥25 mmHg). Lowering of water PO₂ from 150 to 25 mmHg resulted in normoxic fish showing a 3–2 fold increase in V_G. The increase was the result of an elevation in V_{S, R} rather than f_R. Among normoxia acclimated specimens, small fish showed a higher V_G per unit weight than the large ones in both normoxia (PO₂ =150 mmHg) and hypoxia (PO₂ = 15 mmHg). A decrease in the ventilatory requirement (V_G/VO₂) with increased body weight was recorded in hypoxia (PO₂ = 15 mmHg).     

On the conservative nature of the gill filaments of sharks

Synopsis Gill filaments of one highly active and two less active shark species exhibit a conservative morphological scheme including such features as branchial canopies, marginal lamellar projections, and enlarged, discrete outer marginal lamellar channels and lateral lamellar sinuses. The specific spatial orientation of the secondary lamellae respective to one another, the gill filaments, and the interbranchial septa create what appears as one-way interfilament water channels, suggesting the presence of an efficient branchial countercurrent system. It is proposed that the fortified structure of shark gills allows many shark species to ventilate passively without having evolved gill filament modifications as apparently did some highly active teleosts. This in turn may have expedited the evolution of lamnid shark species through pre-adaptation to a swift oceanic lifestyle.