Pharmacology of the retractor lentis muscle of the bluegill (Lepomis macrochirus)

We studied accommodative nerve-muscle transmission in a teleost fish, the bluegill (Lepomis macrochirus). The muscle contracted with electrical stimulation to move the spherical lens along the pupil plane in the eye ball so that the distance between the lens and retina changes. The dose-dependent effect on the lens muscle of acethylcholine and its related substances (nicotine, muscarine, curare and atropine), glutamate, gamma-aminobutyric acid, serotonin, glycine, dopamine and noradrenaline was observed with a binocular microscope. Its video image was processed by a computer to measure the shortening of the muscle. Among the transmitter candidates applied only acethylcholine caused a contraction and elicited the lens movement. Muscarine caused strong contraction of the muscle even in a low concentration (1 nM) while nicotine did not. The contraction was antagonized by atropine but not curare. Previous studies based on intraperitoneally injected reagents suggested a cholinergic innervation on the lens muscle. Our pharmacologic results verified by direct anatomical measurements on the isolated lens-muscle-nerve preparation unambiguously establish the dose-dependent muscarinic-cholinergic innervation to the lens muscle by parasympathetic postganglionic fibers.Abbreviations Ach Acetylcholine - GABA gamma-aminobutyric acid - TTX tetrodotoxin - RML Resting muscle length     

Restricted distribution of laminin α1 chain in normal adult mouse tissues

The distribution of laminin α1 chain in adult mouse tissue was determined by immunofluorescence using monoclonal antibody 200, reacting with the globular carboxyterminus E3 fragment of α1 chain. Strong reactivity was noted only in a few tissues. Reactivity was restricted to epithelial basement membranes. Expression was noted in several epithelial basement membranes of the urinary tract, and male and female reproductive organs. In addition, expression was seen in some parts of the nervous system. Expression was seen in pia mater which surrounds the brain, and in the extracellular matrices covering the vitreous chamber and the lens of the eye. Staining was seen in the adrenal gland cortex, with strongest staining in the zona glomerulosa. Staining was negative in all other studied epithelial basement membranes, such as the lung (trachea or lung epithelium), epidermis, and all parts of the gastrointestinal tract (liver, gut) except for weak staining in the ventricle and Brunner’s glands. No expression was seen in basement membranes of fat, Schwann, or endothelial cells in any studied parts of the body. Both small- and large-size vessel walls were negative both in endothelial basement membranes and blood vessel walls, with the exception of some larger brain blood vessels in locations where epithelial cells have invaginated. Neither smooth muscle, myocardium or striated muscle expressed α1 chain. We conclude that α1-containing heterotrimers including laminin-1 (α1β1γ1) have a very restricted tissue distribution.     

DNA损伤与眼病相关性的研究进展

DNA损伤是复制过程中发生的DNA核苷酸序列永久性改变,并导致遗传特征改变的现象。对DNA损伤与修复的研究是现代分子生物学研究的热点之一。目前,DNA损伤对眼组织细胞凋亡、基因改变、细胞活性的影响已成为眼科疾病研究的热点。在年龄相关性白内障、老年性黄斑病变等眼科疾病的病因研究中证实氧化应激是其主要致病因素,当机体遭受有害刺激导致氧化应激时,机体、组织、细胞受到一系列损伤,而DNA的损伤对氧化应激最为敏感。本文就DNA损伤及其与晶状体上皮细胞及视网膜色素上皮细胞相关性的研究进展作一综述,为眼科疾病的研究以及防治提供新的方法及思路。     

Insulin-like growth factor receptor-1 and nuclear factor κB are crucial survival signals that regulate caspase-3-mediated lens epithelial cell differentiation initiation

It is now known that the function of the caspase family of proteases is not restricted to effectors of programmed cell death. For example, there is a significant non-apoptotic role for caspase-3 in cell differentiation. Our own studies in the developing lens show that caspase-3 is activated downstream of the canonical mitochondrial death pathway to act as a molecular switch in signaling lens cell differentiation. Importantly, for this function, caspase-3 is activated at levels far below those that induce apoptosis. We now have provided evidence that regulation of caspase-3 for its role in differentiation induction is dependent on the insulin-like growth factor-1 receptor (IGF-1R) survival-signaling pathway. IGF-1R executed this regulation of caspase-3 by controlling the expression of molecules in the Bcl-2 and inhibitor of apoptosis protein (IAP) families. This effect of IGF-1R was mediated through NFκB, demonstrated here to function as a crucial downstream effector of IGF-1R. Inhibition of expression or activation of NFκB blocked expression of survival proteins in the Bcl-2 and IAP families and removed controls on the activation state of caspase-3. The high level of caspase-3 activation that resulted from inhibiting this IGF-1R/NFκB signaling pathway redirected cell fate from differentiation toward apoptosis. These results provided the first evidence that the IGF-1R/NFκB cell survival signal is a crucial regulator of the level of caspase-3 activation for its non-apoptotic function in signaling cell differentiation.     

Silencing of sigma-1 receptor induces cell death in human lens cells

Sigma receptors have no known homology with other receptor systems, have no known natural ligands, but appear to play a critical role in a large diversity of cell functions. In the absence of a conventional pharmacology, siRNA technology provides a direct means of elucidating the major cell signaling pathways influenced by this receptor system. The non-transformed human lens cell line FHL124 was found to express the sigma-1 receptor (Sig-1R) and was employed for these studies. 72 h of transfection with either of the two siRNA directed against the sigma-1 receptor reduced messenger RNA and protein levels by over 70 and 60% respectively. Subsequent incubation for 96 h in culture medium (EMEM) supplemented with 5% serum gave a partial recovery of message, but there was no significant increase in protein. LDH leakage assays showed that significant cell death occurred during this time with an increased expression of caspase-3. Thrombin (10 nM) drives the growth of lens cells with a concomitant increase in ERK and Akt phosphorylation. These increases were inhibited in the cells where knockdown had occurred but not in cells exposed to scrambled siRNA. This study establishes a central role for Sig-1R in cell survival and death.     

Differentiation-dependent modification and subcellular distribution of aquaporin-0 suggests multiple functional roles in the rat lens

Using immunohistochemistry and mass spectrometry, differentiation-dependent changes in the subcellular distribution and processing of aquaporin-0 (AQP0) have been mapped in the rat lens. Sections labelled with C-terminal tail AQP0 antibodies yielded two concentric rings of labelling with minimal signal in the lens core. The rings were separated by a transient zone of decreased labelling located prior to the transition of differentiating fiber (DF) cells into mature denucleated fiber (MF) cells. Mass spectrometry showed that the loss of core labelling was due to AQP0 cleavage, while the transient loss of labelling was more likely caused by masking of the antibody epitope. AQP0 subcellular distribution changed with radial distance into the lens. In peripheral DF cells, AQP0 was found throughout both broad and narrow side membranes. In deeper-lying DF cells, AQP0 aggregated into plaque-like structures located on the broad sides. This shift occurred prior to the transient loss of AQP0 signal, and coincided with formation of broad-side membrane invaginations between adjacent fiber cells to which filensin, a known binding partner of AQP0, was also localized. After nuclei loss, AQP0 was once again distributed throughout MF cell membranes. In the absence of protein synthesis, the observed subcellular redistribution of AQP0 in DF and subsequent cleavage of AQP0 in MF are suggestive of a switch in the function of AQP0 from a water channel to a junctional protein.     

Semaphorin3A/neuropilin-1 signaling acts as a molecular switch regulating neural crest migration during cornea development

Cranial neural crest cells migrate into the periocular region and later contribute to various ocular tissues including the cornea, ciliary body and iris. After reaching the eye, they initially pause before migrating over the lens to form the cornea. Interestingly, removal of the lens leads to premature invasion and abnormal differentiation of the cornea. In exploring the molecular mechanisms underlying this effect, we find that semaphorin3A (Sema3A) is expressed in the lens placode and epithelium continuously throughout eye development. Interestingly, neuropilin-1 (Npn-1) is expressed by periocular neural crest but down-regulated, in a manner independent of the lens, by the subpopulation that migrates into the eye and gives rise to the cornea endothelium and stroma. In contrast, Npn-1 expressing neural crest cells remain in the periocular region and contribute to the anterior uvea and ocular blood vessels. Introduction of a peptide that inhibits Sema3A/Npn-1 signaling results in premature entry of neural crest cells over the lens that phenocopies lens ablation. Furthermore, Sema3A inhibits periocular neural crest migration in vitro. Taken together, our data reveal a novel and essential role of Sema3A/Npn-1 signaling in coordinating periocular neural crest migration that is vital for proper ocular development.     

Gdf6a is required for the initiation of dorsal–ventral retinal patterning and lens development

Dorsal–ventral patterning of the vertebrate retina is essential for accurate topographic mapping of retinal ganglion cell (RGC) axons to visual processing centers. Bone morphogenetic protein (Bmp) growth factors regulate dorsal retinal identity in vertebrate models, but the developmental timing of this signaling and the relative roles of individual Bmps remain unclear. In this study, we investigate the functions of two zebrafish Bmps, Gdf6a and Bmp4, during initiation of dorsal retinal identity, and subsequently during lens differentiation. Knockdown of zebrafish Gdf6a blocks initiation of retinal Smad phosphorylation and dorsal marker expression, while knockdown of Bmp4 produces no discernable retinal phenotype. These data, combined with analyses of embryos ectopically expressing Bmps, demonstrate that Gdf6a is necessary and sufficient for initiation of dorsal retinal identity. We note a profound expansion of ventral retinal identity in gdf6a morphants, demonstrating that dorsal BMP signaling antagonizes ventral marker expression. Finally, we demonstrate a role for Gdf6a in non-neural ocular tissues. Knockdown of Gdf6a leads to defects in lens-specific gene expression, and when combined with Bmp signaling inhibitors, disrupts lens fiber cell differentiation. Taken together, these data indicate that Gdf6a initiates dorsal retinal patterning independent of Bmp4, and regulates lens differentiation.     

Activation of Six1 target genes is required for sensory placode formation

In vertebrates, cranial placodes form crucial parts of the sensory nervous system in the head. All cranial placodes arise from a common territory, the preplacodal region, and are identified by the expression of Six1/4 and Eya1/2 genes, which control different aspects of sensory development in invertebrates as well as vertebrates. While So and Eya can induce ectopic eyes in Drosophila, the ability of their vertebrate homologues to induce placodes in non-placodal ectoderm has not been explored. Here we show that Six1 and Eya2 are involved in ectodermal patterning and cooperate to induce preplacodal gene expression, while repressing neural plate and neural crest fates. However, they are not sufficient to induce ectopic sensory placodes in future epidermis. Activation of Six1 target genes is required for expression of preplacodal genes, for normal placode morphology and for placode-specific Pax protein expression. These findings suggest that unlike in the fly where the Pax6 homologue Eyeless acts upstream of Six and Eya, the regulatory relationships between these genes are reversed in early vertebrate placode development.