Vrensen Gijs F J M, van Marle Jan, Jonges Remmet, Voorhout Wim, Breipohl Winrich, Wegener Alfred R
Department of Ophthalmology, Leiden University Medical Centre, Leiden, The Netherlands.
Exp Eye Res. 2004 Mar;78(3):661-72. doi: 10.1016/j.exer.2003.07.004.
Tryptophan deficiency is known for long time to cause cataract in rats. However, up till now the underlying mechanism is still enigmatic. Histological studies showed an extended lens bow suggesting that the normal breakdown of nuclei in the lens fibres is arrested under these conditions. Using advanced ultrastructural techniques we aimed to clarify this aberrant final differentiation of lens fibres. Albino and pigmented rats were permanently or intermittently raised on a tryptophan deficient diet for 12 and 16 weeks, respectively. Rats of the same age raised on a normal diet served as controls. Lenses were treated for light and electron microscopy. For histology sections were stained for DNA and gamma-crystallins. In addition to routine transmission electron microscopy (TEM), ultrathin sections were subjected to electron tomography and energy dispersive X-ray microanalysis (EDX). Histology verified the extended lens bow for albino and pigmented rats and showed that in the intermittent period of normal diet the fibre nuclei are broken down as in controls. It was further shown that gamma-crystallins are co-localized with DNA in the nuclear domain. TEM revealed that during final differentiation nuclear chromatin becomes highly compacted in a chromosome-like manner and than rapidly evanesces in control rats. This compacted stage persists indefinitely in the tryptophan deficient rats. Electron tomography showed that during differentiation chromatin is first uncoiled to 30 nm solenoids, subsequently to highly compacted 10 nm beads-on-a-string fibrils and than is segregated from the nuclear proteins. EDX revealed that the late stage persisting nuclei consist of domains rich in DNA associated with histones and in domains with mainly proteins. This study corroborates previous findings on the final breakdown of nuclei of lens fibres. It further shows that the chromatin is ultimately uncoiled to beads-on-a-string fibrils and that as the last step chromatin is broken down at this unmasked stage. Except for this last step nuclear breakdown is identical in control and tryptophan deficient rats suggesting that it is not the availability of tryptophan for protein synthesis in general which causes the arrest. Two alternatives for this final arrest are discussed. A low tryptophan content, most pronounced in deeper cortical layers, may inhibit the late synthesis of the DNases and proteases necessary for chromatin breakdown. The radical scavenging by indoleamine 2,3-dioxygenase, which cleaves the pyrrole ring of tryptophan to form formylkynurenine using free oxygen radicals, is impaired by low levels of tryptophan. This decreased scavenging of oxygen radicals will expose the catalytic enzymes for chromatin breakdown, residing in the nucleus in an inactive form for quite a long period, to high levels of oxygen radicals and may affect the activity of these enzymes and therefore the execution of the chromatin breakdown.
长期以来，人们已知色氨酸缺乏会导致大鼠患白内障。然而，迄今为止其潜在机制仍然不明。组织学研究显示晶状体弓延长，这表明在这些条件下晶状体纤维细胞核的正常解体被阻止。我们使用先进的超微结构技术旨在阐明晶状体纤维这种异常的最终分化。分别将白化病大鼠和有色大鼠长期或间歇性地饲养在色氨酸缺乏的饮食中12周和16周。以正常饮食饲养的同年龄大鼠作为对照。对晶状体进行光镜和电镜处理。对于组织学，切片用DNA和γ-晶状体蛋白染色。除了常规透射电子显微镜（TEM）外，超薄切片还进行了电子断层扫描和能量色散X射线微分析（EDX）。组织学证实了白化病大鼠和有色大鼠的晶状体弓延长，并表明在正常饮食的间歇期，纤维细胞核如对照组一样解体。进一步表明γ-晶状体蛋白与核区域中的DNA共定位。TEM显示，在最终分化过程中，对照大鼠的核染色质以类似染色体的方式高度浓缩，然后迅速消失。在色氨酸缺乏的大鼠中，这种浓缩阶段无限期持续。电子断层扫描显示，在分化过程中，染色质首先解旋成30nm的螺线管，随后解旋成高度浓缩的10nm串珠状纤维，然后与核蛋白分离。EDX显示，后期持续存在的细胞核由富含与组蛋白相关的DNA的区域和主要由蛋白质组成的区域组成。这项研究证实了先前关于晶状体纤维细胞核最终解体的发现。它进一步表明，染色质最终解旋成串珠状纤维，并且作为最后一步，染色质在这个暴露阶段被分解。除了这最后一步，对照大鼠和色氨酸缺乏大鼠的核解体是相同的，这表明一般而言不是色氨酸用于蛋白质合成的可用性导致了停滞。讨论了这种最终停滞的两种可能性。色氨酸含量低，在更深的皮质层中最为明显，可能会抑制染色质分解所需的DNase和蛋白酶的后期合成。色氨酸水平低会损害吲哚胺2,3-双加氧酶的自由基清除作用，该酶利用自由基切割色氨酸的吡咯环以形成甲酰犬尿氨酸。这种自由基清除作用的降低将使长期以无活性形式存在于细胞核中的染色质分解催化酶暴露于高水平的自由基中，并可能影响这些酶的活性，从而影响染色质分解的执行。
