人眼晶状体醛脱氢酶1的克隆表达和酶特性研究

目的:研究人眼ALDH1A1的结构与功能学特点。方法:使用标准的PCR技术,从人晶状体cDNA文库扩增ALDH1A1。扩增的ALDH1A1(1.5kb)被插入到克隆表达载体pET-28b,再转染到菌株BL21DE3中。表达出来的蛋白质经His-tag柱纯化,使用thrombin切除His-tag。对纯化的ALDH1A1的酶学特点,如酶活性与辅酶、缓冲液、还原剂、pH以及析出方式等的关系进行了检测。结果:从人晶状体的基因文库,成功扩增了ALDH1A1,重组的人晶状体ALDH1A1为1506bp的DNA及501个氨基酸组成,分子量为54.8kDa,PI=6.8。与人的肝脏AL-DH1A1100%同源,与大鼠和小鼠肝脏的ALDH1A1有85%的同源性。Thrombin酶切较imidazole洗脱的ALDH1A1活性保持得更持久,纯化的ALDH1A1在碱性的sodiumpyrophosphate反应缓冲液中,NAD为辅酶和dTT作为还原剂时的活性较高,其活性随pH增高而增加。结论:ALDH1A1与人肝组织的同工酶有相似的作用。     

Molecular cloning of three zebrafish lin7 genes and their expression patterns in the retina

The vertebrate retina develops from an undifferentiated sheet of neuroepithelial cells, whose differentiation requires the generation and maintenance of the correct cellular polarity. To examine the role of cell polarity in retinal development, we cloned three zebrafish lin7 genes (lin7a, lin7b, and lin7c), which each encodes a protein candidate that is required for generation/maintenance of neuroepithelial cell junctions. These three zebrafish Lin7 proteins share over 78% amino acid identity and contain both L27 and PDZ domains that are present in all Lin7 homologs. Immunoblots revealed that the Lin7b and Lin7c proteins were first expressed in the developing eye by 24hr postfertilization (hpf), while Lin7a was not detected in the eye until 72 hpf. At 33 hpf, the Lin7 proteins localized at, or slightly apical of, the actin-associated adherens junctions in the retinal neuroepithelium. This subcellular distribution required the expression of the Nok protein. In the absence of Nok, the Lin7 proteins failed to localize to either the ectopic adherens junctions or the cell membrane. At 4 days postfertilization, in situ hybridisation revealed that all three lin7 genes were expressed in both the ganglion cell layer and the bipolar cell region of the inner nuclear layer. The lin7a gene was also expressed in the amacrine and horizontal cell regions of the inner nuclear layer, while lin7c was also expressed in the outer nuclear layer. In the adult retina, where Lin7a is the predominant form expressed, the Lin7 proteins were localized to the outer and inner plexiform layers, the bipolar and horizontal cells of the inner nuclear layer, and the ganglion cells. These results suggest that the three zebrafish Lin7 proteins possess partially redundant, yet essential, roles in retinal development.     

Molecular cloning and complete nucleotide sequence of the cDNA encoding a bovine lens intrinsic membrane protein (MP19)

Recently, we reported the partial characterization of bovine lens intrinsic membrane proteins having apparent SDS-PAGE derived molecular mass of 19, 21, and 23 kDa, and determined that they contained identical NH2- terminal amino acid sequences for the first 20 amino acids. From this amino acid sequence information, a mixed synthetic oligonucleotide was constructed and used to screen a calf lens lambda gt11 cDNA library in order to isolate and characterize the cDNA coding for this membrane polypeptide(s). Two separate cDNA clones were isolated and sequenced, and were found to have an identical sequence of 883 bases with an open reading frame coding for a polypeptide of 173 amino acids, having a molecular mass of 19,683 Daltons. The first 20 amino acids of the translated sequence were identical to that determined by our laboratory previously, and the last seven amino acids were identical to that recently determined by another laboratory from analysis of the extracted polypeptides, indicating that this cDNA is the authentic molecule coding for MP19.     

Deletion of the X-linked opsin gene array locus control region (LCR) results in disruption of the cone mosaic

Blue cone monochromacy (BCM) is an X-linked condition in which long- (L) and middle- (M) wavelength-sensitive cone function is absent. Due to the X-linked nature of the condition, female carriers are spared from a full manifestation of the associated defects but can show visual symptoms, including abnormal cone electroretinograms. Here we imaged the cone mosaic in four females carrying an L/M array with deletion of the locus control region, resulting in an absence of L/M opsin gene expression (effectively acting as a cone opsin knockout). On average, they had cone mosaics with reduced density and disrupted organization compared to normal trichromats. This suggests that the absence of opsin in a subset of cones results in their early degeneration, with X-inactivation the likely mechanism underlying phenotypic variability in BCM carriers.     

Identification and characterization of a retina-specific calpain (Rt88) from rat

PURPOSE: To identify and characterize a newly discovered calpain termed Rt88 from rat retina. METHODS: Rt88 in retina under normal physiological conditions was characterized in Sprague-Dawley rats of various ages by competitive RT-PCR, Northern blot analysis, cDNA cloning and sequencing. Recombinant Rt88 was expressed in the baculovirus system and characterized by casein zymography and immunoblotting. RESULTS: Rt88 was sequenced and found to be similar to muscle calpain p94 except for three differences. A different exon 1 (as in lens Lp82 calpain) was present, and exons 15 and 16 in the unique IS2 region of muscle p94 were deleted. Of eleven tissues studied, mRNA for Rt88 was found only in retina where Rt88 increased with maturation and then remained constant. Casein zymography showed that rRt88 was proteolytically active after activation by calcium, but intact rRt88 was rapidly broken due to the presence of the IS1 region in domain II. CONCLUSIONS: Rt88 is a retina-specific, calcium activated protease from the calpain superfamily (EC 3.4.22.17) of cysteine proteases. Rt88 is a recently identified member of the AX1 subfamily of calpains showing alternative exon 1 usage. So far, all AX1 subfamily members are from eye. Rt88 may perform specific proteolytic functions during development, normal turnover, or pathological degeneration of retinal proteins.     

Erratum: Temporal and spatial patterns of opsin gene expression in the zebrafish (Danio rerio): corrections with additions.

We report here a reexamination of the developmental expression of cone opsins in the zebrafish retina. The red- and blue-sensitive opsins appear at 51 h postfertilization (hpf) whereas ultraviolet (UV) opsin is not seen until after 55 hpf. More cells show red cone opsin expression than blue at 51 and 55 hpf, indicating the sequence of cone opsin expression in zebrafish is first red, then blue, and finally UV. Curiously, morphological development of the cones is in reverse order; UV cones appear quite mature by day 6-7 postfertilization (pf), but morphologically, red cones do not appear adult-like until 15-20 days pf.     

Identification and cloning of the Na/HCO(3-) cotransporter (NBC) in human corneal endothelium

Fluid secretion by the corneal endothelium is associated with the net flux of HCO(3)(-) from basolateral (stromal) to apical (anterior chamber) sides of the tissue. In this study we asked if Na(+)/HCO(3)(-) cotransporter (NBC-1) protein expression and functional activity are present in freshly isolated human corneal endothelium. Immunoblot analysis using a polyclonal antibody to NBC-1 showed a single band at approximately 130 kDa. Indirect immunofluorescence indicated that NBC-1 is expressed on the basolateral, but not apical side of human corneal endothelium. RT-PCR was used to determine whether the kidney or pancreatic isoform of NBC-1 is expressed. Using the specific primers for pNBC and kNBC isoforms, RT-PCR showed that only pNBC could be detected in human corneal endothelium. The product was cloned and confirmed by sequencing. Full-length NBC-1 was also cloned from human corneal endothelium. This clone (hcNBC) is 100% identical to the longer, more common form of NBC [pNBC; 1079 amino acids (aa); 122 kDa in human heart, pancreas and prostate]. To test for functional activity of NBC-1, freshly isolated endothelium was loaded with the pH sensitive fluorescent dye BCECF and HCO(3)(-) fluxes were measured. HCO(3)(-) fluxes were Na(+)-dependent, electrogenic and H(2)-DIDS sensitive. We conclude that the long isoform of the sodium bicarbonate cotransporter (pNBC-1) is expressed on the basolateral side of fresh human corneal endothelium (hcNBC). The shorter form, kNBC, could not be detected. As in bovine corneal endothelium, hcNBC is instrumental in loading HCO(3)(-) into endothelial cells from the basolateral membrane.     

Molecular cloning of the bovine MYOC and induction of its expression in trabecular meshwork cells

PURPOSE: Myocilin gene (MYOC) was identified as one of the disease-causing genes of primary open-angle glaucoma. This study was conducted to establish a system for the investigation of the biological role of MYOC in vitro by using bovine eyes, which are easy to obtain and have been widely used to examine the aqueous outflow system. The cDNA sequence of the bovine MYOC was determined and its expression in bovine eyes was examined with a quantitative polymerase chain reaction (PCR) assay. METHODS: Bovine MYOC cDNA was obtained from cultured bovine trabecular meshwork cells, and part of its sequence was determined using a primer pair designed based on the known sequence of the human MYOC gene. The 3' and 5' ends of this sequence were determined using the method of 3' and 5' rapid amplification of cDNA ends. The induction of the MYOC gene in cultured bovine trabecular meshwork cells after exposure to dexamethasone was quantitatively examined with real-time quantitative PCR using a probe designed according to the sequence of the determined bovine MYOC gene. RESULTS: Bovine MYOC protein was composed of 490 amino acids, which was 81.6% identical with that of human MYOC protein. Most of the amino acid residues of which mutation was reported to cause glaucoma were conserved in the bovine MYOC protein. After 2 weeks of treatment with 500 nM dexamethasone, expression of bovine MYOC mRNA was amplified 14-fold (14.1+/-5.1-fold, mean +/- SEM) measured by real-time quantitative PCR. CONCLUSIONS: The cDNA sequence of the bovine MYOC gene had a high degree of similarity to that of the human MYOC gene. Investigation of the function of bovine MYOC may contribute to identifying the role of MYOC protein in the aqueous outflow system.     

Purification and characterization of lens specific calpain (Lp82) from bovine lens.

Ubiquitous type m-calpain and lens specific Lp82 calpain were separated and partially purified from fetal bovine lens and the enzymatic characteristics were compared. Lens m-calpain required 200 microM calcium for 1/2 maximal activity, while Lp82 required 30 microM. Both types of calpains were inhibited by 0.1 mM E64, and 5 mM iodoacetamide, but not by 1 mM phenylmethylsulfonyl fluoride. Lp82 was insensitive to 1 microM calpastatin peptide while m-calpain was effectively inhibited. In the presence of calcium, m-calpain lost most of its activity within 2 hr, while Lp82 was continually active for 18 hr. Both calpains cleaved the natural substrates betaA3 and alphaB crystallins in a similar manner. However, incubation of alphaA crystallin with m-calpain removed ten amino acid residues from its C-terminus, while incubation with Lp82 removed only five residues. The latter truncation product of alphaA was also found in vivo. These data suggested that Lp82 may have a more important role than m-calpain in modification of crystallins during lens maturation.     

Partial re-incorporation of corner cones in the retina of the Atlantic salmon (Salmo salar)

The distribution of corner (putative ultraviolet-sensitive) cones in the retina of Atlantic salmon was examined from the small juvenile (parr) stage to the adult stage (approaching sexual maturation). Small parr weighing approximately 5 g lacked corner cones everywhere except, mainly, near the dorsal periphery. Large fish ( approximately 5 kg) approaching sexual maturation showed corner cones in other areas of the dorsal retina besides the periphery. These areas, characterized by low resolving power, had similar corner cone densities to analogous areas in the smolt retina, suggesting that corner cones are formed in the periphery and incorporated into the dorsal retina of the Atlantic salmon sometime during the smolt stage. This incorporation is partial both in numbers of cones and in location (only the dorsal retina is affected). These findings contrast with the situation in rainbow trout where corner cones from existing mosaics are only partially lost from the ventral retina, if at all, and where production and incorporation of these cones into the dorsal retina occurs throughout life. Thus, in salmonids, there are at least two different strategies that determine retinal corner cone distributions.     

Generation, characterization, and molecular cloning of the Noerg-1 mutation of rhodopsin in the mouse

We performed genome-wide mutagenesis of C57BL/6J mice using the mutagen N-ethyl-N-nitrosourea (ENU) and screened the third generation (G3) offspring for visual system alterations using electroretinography and fundus photography. Several mice in one pedigree showed characteristics of retinal degeneration when tested at 12-14 weeks of age: no recordable electroretinogram (ERG), attenuation of retinal vessels, and speckled pigmentation of the fundus. Histological studies showed that the retinas undergo a photoreceptor degeneration with apoptotic loss of outer nuclear layer nuclei but visual acuity measured using the optomotor response under photopic conditions persists in spite of considerable photoreceptor loss. The Noerg-1 mutation showed an autosomal dominant pattern of inheritance in progeny. Studies in early postnatal mice showed degeneration to occur after formation of partially functional rods. The Noerg-1 mutation was mapped genetically to chromosome 6 by crossing C57BL/6J mutants with DBA/2J or BALB/cJ mice to produce an N2 generation and then determining the ERG phenotypes and the genotypes of the N2 offspring at multiple loci using SSLP and SNP markers. Fine mapping was accomplished with a set of closely spaced markers. A non-recombinant region from 112.8 Mb to 115.1 Mb was identified, encompassing the rhodopsin (Rho) coding region. A single nucleotide transition from G to A was found in the Rho gene that is predicted to result in a substitution of Tyr for Cys at position 110, in an intradiscal loop. This mutation has been found in patients with autosomal dominant retinitis pigmentosa (RP) and results in misfolding of rhodopsin expressed in vitro. Thus, ENU mutagenesis is capable of replicating mutations that occur in human patients and is useful for generating de novo models of human inherited eye disease. Furthermore, the availability of the mouse genomic sequence and extensive DNA polymorphisms made the rapid identification of this gene possible, demonstrating that the use of ENU-induced mutations for functional gene identification is now practical for individual laboratories.     

Immunochemical characterization of the major low molecular weight polypeptide (10K) from human cataractous lenses

Polyclonal antisera to whole crystallins and to synthetic peptides corresponding to various sequences of these crystallins have been used to probe Western blots that contain a low molecular weight component of approximately 10,000 daltons found in the water-soluble fractions from human cataractous lenses. This 10K component binds only to antiserum made against human gamma crystallin. Incubation of human cataractous lens homogenates with alpha chymotrypsin or trypsin will produce low molecular components of similar molecular weight, and identical specificity of binding to the gamma crystallin antiserum. Together, these results suggest that the gamma crystallins constitute a class of macromolecules that are susceptible to in vivo proteolysis during cataractogenesis of the aged human lens.     

Myopia and personality: the genes in myopia (GEM) personality study

PURPOSE: A long-held view among the medical and broader community is that people who are short-sighted (myopic persons) have distinctive personality characteristics such as introversion and conscientiousness. However, existing research on this question is flawed, and its findings are inconsistent. The authors therefore aimed to determine whether myopia and personality are associated. METHODS: The authors examined twins recruited through the Australian Twin Registry and a clinical-based family sample through a proband from a Melbourne Excimer Laser Clinic. There was no relation between family members and twins recruited in our study. Each individual underwent a full eye examination, completed a standard medical and general questionnaire, and was administered a five-factor model International Personality Item Pool (IPIP) inventory (Openness, Conscientiousness, Extroversion, Agreeableness, Neuroticism). Myopia was defined as worse than or equal to -0.50 (DS) spherical equivalent in the eye with the least refractive error. RESULTS: Data from 633 individual twins aged 18 to 83 years (mean, 53.04 years) and 278 family members aged 11 to 90 years (mean, 49.84 years) were analyzed. Prevalence of myopia was 35.7% for twins and 47.6% for family members. Mean spherical equivalent was +0.13 DS (95% CI, +/-0.16) for twins and -1.13 DS (95% CI, +/-0.25) for family members. Correlation and regression results for personality for both sample cohorts after multivariate analysis did not support the view that myopic persons are introverted or conscientious; however, there was a significant but small association between myopia and Agreeableness (r = 0.08, P < 0.05). In multivariate analysis with age, sex, education, and the five personality factors entered as predictors, Openness was the only significant personality predictor of myopia in both samples. CONCLUSIONS: This is the first multivariate study to assess links between personality and myopia using the IPIP. The long-held view that myopic persons are introverted and conscientious may reflect intelligence-related stereotypes rather than real correlations. Furthermore, the predictive characteristic of intellect, subsumed in Openness, appeared to be representative of a previously reported link between intellective abilities (IQ) and myopia rather than personality and myopia.     

Lims A:一个新的LIM同源域基因的克隆和初步分析

目的:克隆与分析大鼠不同剪切的Lims基因。方法:应用巢式RT-PCR,以SD大鼠cDNA为模板,扩增Lims基因不同剪切子,构入PinPointTMXa-1T质粒,测序鉴定。结果:测序表明克隆了新的Lims基因变异剪切体Lims A,该变异剪切体编码区为1014bp,编码338个氨基酸。结论:比较基因组学分析显示,成功地克隆了一新的大鼠Lims基因剪切子Lims A,为进一步探索Lims基因在增生性玻璃体视网膜病变中的功能奠定基础。