反义PCNA与反义bcl-2脱氧寡核苷酸抑制人视网膜色素上皮生长

目的　观察反义增生细胞核抗原 (PCNA)和反义 bcl-2脱氧寡核苷酸对培养的人视网膜色素上皮细胞 (RPE)生长的作用 .方法　用 DNA合成仪合成有 1 8个核苷酸的反义PCNA和 1 5个核苷酸的反义 bcl- 2脱氧寡核苷酸片段 ,以不同浓度加入 RPE培养液 ,培养 2～ 4d,用 SABC法检测细胞PCNA和 bcl- 2的表达 ,用 MTT法及流式细胞仪测定细胞抑制率 .结果　 6 .2 5～ 1 0 0μm ol· L- 1 的反义 PCNA对 RPE的抑制率为 8.3%～ 38.4% ,呈剂量依赖性 ,并抑制细胞 PCNA的表达 .反义 bcl- 2未显示抑制作用 .结论　反义 PCNA脱氧寡核苷酸可抑制近 40 %的 RPE生长 ,其临床应用值得进一步研究     

Leptomeningeal lipid storage patterns in Fabry disease

We found two patterns of leptomeningeal storage that reflect two basic visceral storage patterns in Fabry disease. (i) A generalized-type leptomeningeal storage pattern, affecting all main leptomeningeal cell types (external arachnoideal epithelium, fibroblasts, vessel wall elements), was a consistent finding in three cases of classical generalized visceral phenotype. (ii) A localized leptomeningeal storage pattern was expressed, to a high degree, solely in the external arachnoidal epithelium; this pattern was found in one case with the variant visceral-restricted-type storage (confined to the cardiocytes). Thus, the external arachnoidal epithelium may be particurlarly susceptible to Fabry lipid storage, probably caused by a distinctly larger sustained lysosomal lipid load as compared to other cell types.     

Effects of prolonged uniocular dark adaptation on the direct-current electroretinogram of pigmented and albino rabbits

The direct-current electroretinogram of seven pigmented and seven albino rabbits was recorded from both eyes for almost 4 h in response to repeated identical light stimuli. Stimulus duration was 10 s, light intensity was 6.8 × 10² lux, and the interval between the beginning of succeeding light stimuli was 3 min. The dark-adaptation period preceding light stimulation was 30 min for one of the eyes (unoccluded eye) and 150 min for the contralateral eye (occluded eye), which was patched during the first part (117 min) of the experiment. In pigmented animals, the b- and c-wave amplitudes of the unoccluded eye slowly increased during the first part of the experiment but not significantly during the second. The a-wave amplitude was not significantly changed. After removal of the cover, the a- and b-wave amplitudes of the occluded eye immediately attained but did not exceed the level of those in the unoccluded eye, irrespective of the light adaptation induced by the stimulus flashes previously presented to the unoccluded eye. (Control experiments on six pigmented rabbits confirmed that stimuli identical to those used in the main part of the study caused a light adaptation, since a decrease in a- and b-wave amplitudes occurred after the first light stimulus following an initial dark-adaptation period of 2 h for both eyes.) In albino rabbits, electroretinogram responses were clearly discernible in the occluded eye also during the first part of the experiment, probably because of transillumination of the head. In other respects, the results were essentially similar to those of pigmented animals. The observation that occluded eyes did not dark adapt better, as judged by the electroretinogram responses, than contralateral eyes given repeated light adaptive stimuli may indicate the presence of a mechanism for transfer of adaptation information between the eyes.     

A cyclic adenosine monophosphate agonist elevates the b- and c-waves of the rabbit direct-current electroretinogram

The effects of the stable cyclic adenosine monophosphate analogue adenosine 3, 5-cyclic monophosphorothioate Sp-isomer (Sp-cAMPS) on the direct-current electroretinogram and the standing potential of the eye were studied. Corneal recordings were obtained from unilaterally vitrectomized albino rabbit eyes during alternating intravitreal perfusions with Sp-cAMPS and a control solution (Pharmacia eye irrigating solution). The contralateral eye was used as a control. To evaluate further the effects on the c-wave,in vivo intraretinal microelectrode measurements were made during simultaneous intravitreal perfusion of Sp-cAMPS and irrigating solution, respectively. Sp-cAMPS in concentrations of 1, 10 and 100µM was tested by corneal direct-current electroretinography. There was no significant effect on the a-wave amplitude. The b-wave amplitude was reversibly elevated at an Sp-cAMPS concentration of 100µM (p<0.01, n=7). The c-wave amplitude was reversibly elevated at a concentration of 10µM (p<0.001, n=8), and this effect was more pronounced at 100µM (p<0.001, n=7). The SP increased reversibly at a concentration of 100µM (p<0.001, n=7). Microelectrode recordings were performed with Sp-cAMPS at a concentration of 100µM. The recordings showed significant increases in both the transepithelial potential (p<0.01, n=3) and the slow PIII (p<0.01, n=3). The effects of Sp-cAMPS on the b-wave as well as on the two components of the c-wave suggest influences on both the inner retina and the retinal pigment epithelium of the rabbit eye.Abbreviations PHS Pharmacia eye irrigating solution - AMP adenosine monophosphate - Sp-cAMPS adenosine 3, 5 - cyclic monophosphorothioate Sp-isomer     

Immunolocalization of CD1d in human intestinal epithelial cells and identification of a beta2-microglobulin-associated form

In order to better understand the role of intestinal CD1d, we sought to define the cellular localization and further characterize the biochemical structure of CD1d in human intestinal epithelial cells (IEC). Using a CD1d-specific rabbit anti-gst-CD1d antibody, immunoprecipitation of radiolabeled cell surface proteins detected a previously identified 37 kDa protein as well as a 48-50 kDa protein which were confirmed by Western blotting with a CD1d-specific mAb, D5. Immunoprecipitation of protein lysates with the CD1d-specific mAb, D5 and 51.1.3, and the beta2-microglobulin (beta2m)-specific mAb, BBM.1, followed by N-glycanase digestion and Western blotting with the D5 mAb showed that the 48-50 kDa protein was a beta2m-associated, CD1d glycoprotein. CD1d was immunolocalized to the apical and lateral regions of native small and large intestinal IEC as defined by confocal laser microscopy using the D5 mAb and the rabbit anti-gst-CD1d antibody. In addition, a large apical intracellular pool of CD1d was identified. Identical observations were made with polarized T84 cells. Selective biotin labeling of apical and basolateral cell surfaces followed by immunoprecipitation with the D5 mAb, N-glycanase digestion and avidin blotting confirmed the presence of glycosylated CD1d on both cell surfaces and immunolocalization of the 37 kDa non-glycosylated form of CD1d to the apical cell surface. These studies show that CD1d is located in an ideal position for luminal antigen sampling and presentation to subjacent intraepithelial lymphocytes.     

Degradation of bradykinin by bovine tracheal epithelium and isolated epithelial cells

1. The degradation of bradykinin (BK) labelled with tritiated proline at positions 2 and 3 ([³H]-BK) was determined on the luminal surface of bovine tracheal epithelium, in supernatants obtained from incubations of the luminal tracheal surface, and in suspensions of isolated tracheal epithelial cells. Peptidase inhibitors and identification of peptide fragments were used for characterization of the metabolic pathways.
2. On the luminal surface of intact bovine trachea, [³H]-BK was degraded with a half life of 12.8 min. [1-7]-BK and [1-5]-BK were the major direct metabolites which were further degraded via [1-3]-BK and [2-3]-BK to proline. Metabolism of [³H]-BK was unaltered in the presence of ramiprilat (250 nM) or phosphoramidon (10 μM). Phenanthroline diminished the formation of [1-7]- and [1-5]-BK and abolished the generation of proline.
3. Supernatants obtained from incubations of tracheal epithelium contained kininase activities which steadily increased when tracheae were incubated for longer than 30 min. After 60 min contact with epithelium, the incubation medium contained higher kininase activities than the epithelium itself. The spectrum of kinin metabolites generated by kininases in the supernatant was comparable to that formed by intact epithelium.
4. In suspensions of isolated epithelial cells, [³H]-BK was degraded with a half life of 70 min. The metabolites [1-3]- and [2-3]-BK were formed in parallel to [1-7]- and [1-5]-BK; however, proline was not generated. Degradation of [³H]-BK was not influenced by ramiprilat, but was inhibited by 85% in the presence of phosphoramidon. Phosporamidon markedly inhibited the generation of [1-7]- and [1-5]-BK and nearly abolished the formation of [1-3]- and [2-3]-BK.
5. In conclusion, angiotensin I-converting enzyme and neutral endopeptidase 24.11 are not significantly involved in [³H]-BK degradation on the luminal side of intact tracheal epithelium. The spectrum of metabolites found may in fact reflect the combined activities of metalloendopeptidase 24.15 and post-proline cleaving enzyme. Enzymes showing similar kininase activities are also released from the epithelium. Isolated epithelial cells contain low activities of these kininases, but a high activity of neutral endopeptidases, which may reflect an exclusively basolateral localization of the latter.

     

Pentose shunt activity in developing chick retina and pigment epithelium: A switch in biochemical expression in cultured pigment epithelial cells

Pentose shunt activity in developing chick retina and pigment epithelium was studied by measuring the rate of ¹⁴CO₂ evolution from glucose selectively labelled in the C-1 and C-6 positions. In the retina, shunt activity declines from appreciable levels at stages 29–31 to minimal activity in the 2-week-old hatched chick. Overall retinal metabolism also declines up to stage 45, but dramatically increases again after hatching. Developing chick pigment epithelium has minimal shunt activity at all stages studied. In contrast, cultured chick pigment epithelium has appreciable shunt activity which is constant over a period of several weeks in culture. This appears to be a switch in biochemical differentiation which could form the basis at least in part for subsequent changes in cell types observed in cultured pigment epithelial cells byEguchi and Okada (1973).     

Electron microscopy and X-ray microanalyses of uterine epithelium from lead-injected mice in an experimental delay of implantation

Mice in experimental delay of implantation were injected intravenously with 75 g · g^–1 body weight of lead chloride, corresponding to a dose of lead of about 56 g · g^–1 body weight. Delay of implantation was obtained by ovariectomy 3 days after mating followed by a depot dose of progesterone every fifth day. Electron microscopy showed that the uterine lumen, which was closed in control mice, was opened in lead-injected mice. This morphology suggested that lead caused an increase in uterine secretion. X-ray microanalysis of pyroantimonate precipitates in the uterine epithelium of injected mice demonstrated lead in the precipitates, suggesting that lead could have a direct effect on the function of the uterine epithelium and that lead also could be secreted into the uterine lumen and affect the blastocysts.     

On the Life Span of Olfactory Receptor Neurons

The life span of olfactory receptor neurons was investigated after injection of a retrograde tracer into the olfactory bulb. Mice were injected unilaterally with colloidal gold conjugated with Concanavalin A and their olfactory epithelia were examined after 7, 14, 30, 60, and 90 days. Gold particles could be seen in the epithelia at all survival periods after silver intensification. There was no gold in the epithelia on the uninjected side. In order to test whether gold could be recycled within the epithelium upon the death of receptor neurons, the olfactory bulbs of some mice were ablated 4 days after colloidal gold injection. None of the receptor neurons in these epithelia contained gold at any survival period. To investigate whether gold was continuously available at the injection site, olfactory bulbs were examined by electron microscopy. By 7 days after injection all gold was sequestered intracellularly and was presumably unavailable for uptake by the olfactory axons. These results indicate that olfactory receptor neurons live for at least three times the commonly accepted life span of 30 days. A long life span challenges the widely held view that olfactory receptor neurons are regularly replaced.     

Renal epithelium: reversal of cytotoxic damage by addition of anti-thymocyte globulin

A novel in vitro assay of renal epithelium tight junction function was used to assess the efficacy with which rabbit anti-thymocyte globulin (ATG) blocks epithelium damage mediated by lymphokine-activated killer (LAK) cells. It was found that LAK cells lysed renal epithelial cells poorly in standard chromium-release assays but that they caused a rapid, and almost total, reduction in trans-epithelium monolayer resistance, indicating tight junction failure and, hence, loss of tissue function. LAK cell-mediated cytolysis of the sensitive K562 cell line was completely blocked in the presence of ATG at a concentration of 200 g/ml. Addition of ATG at this concentration to damaged renal cell monolayers in the presence of LAK cells allowed the trans-monolayer resistance to recover rapidly to levels approaching the values recorded before initial addition of LAK cells. On this basis it seems likely that the rapid restoration of renal function frequently observed after appropriate rescue therapy during episodes of acute rejection may reflect subtle changes in tissue function rather than recovery from widespread graft cell cytolysis.