Primary intermediates of rhodopsin studied by low temperature spectrophotometry and laser photolysis. Bathorhodopsin, hypsorhodopsin and photorhodopsin

The primary photochemical processes of rhodopsin studied by low temperature spectrophotometry and picosecond laser spectroscopy in our group was summarized. Low temperature spectroscopic experiments demonstrated that the retinylidene chromophores of hypso- and bathorhodopsins are in a twisted all-trans forms. Excitation of rhodopsin with 532 nm laser pulse (width: 25 psec) yielded a new bathochromic photoproduct "photorhodopsin"; its spectrum was located at longer wavelengths than that of bathorhodopsin. Photorhodopsin decays to bathorhodopsin with time constants of about 200 psec in squid and 40 psec in cattle. Squid and octopus hypsorhodopsins were produced within 25 psec by high energy pulse, but not by low energy pulse. Thus hypsorhodopsin is produced by two photon reactions (sequential two photochemical reactions) and decayed to bathorhodopsin with time constant of 125 psec.     

视网膜光化学损伤大鼠视紫红质基因mRNA表达水平的原位杂交技术检测

目的：研究视网膜光化学损伤状况下视紫红质基因mRNA表达水平的变化规律及其与形态学改变的关系。 方法：应用原位杂交和电镜技术对光化学损伤大鼠视网膜视紫红质基因mRNA表达情况及视网膜形态学损伤性改变进行动态观察。 结果：视紫红质mRNA原位杂交信号主要分布于视网膜光感受细胞层,尤其是其内段和外段;随连续光照时间的延续,其表达迅速下降,且先于视网膜形态学的损伤性改变;同一视网膜中,上方及后极部杂交信号的减弱较下方和周边部更为明显。 结论：视紫红质基因mRNA表达水平降低可能为光化学性视网膜损伤的早期信号。 （中华眼底病杂志,1997,13：228-230）     

视网膜色素变性家系的产前分子诊断

目的：对1例常染色体显性遗传视网膜色素变性家系患者所怀胎儿进行产前分子诊断。方法：应用聚合酶链反应（PCR）和直接测序技术法，对一常染色体显性遗传视网膜色素变性（ADRP）家系成员的所有现存人员的视紫红质基因的外显子进行测序分析。在采用STR位点分析方法排除母体基因组DNA污染后，应用DNA序列测定对胎儿-羊水基因组DNA进行分析。结果：该家系的25名成员中12名患者有视紫红质基因（rhodopsin，RHO）的512C〉T（P171L）突变，均呈杂合子，该错义突变使密码子171由CCA变成CTA。而未受累者的视紫红质基因表现为野生型。对该家系成员进行产前诊断，发现胎儿具有同种致病性突变。结论：视紫红质基因RHO的一种已知突变512C〉T（P171L）是该家系的病因，胎儿带P171L突变，产前诊断和早期干预能避免视网膜色素变性患儿的出生。     

A new microspectrophotometric method for measuring absorbance of rat photoreceptors

A method has been developed for obtaining visual pigment absorbance spectra from fixed, frozen sections of the albino rat retina. The retinas are transversely sectioned at various thicknesses on a cryo-microtome and the resulting slices are examined with a photon-counting microspectrophotometer. Problems caused by the limited pigment content of small receptors are reduced and measurements can be made at well-specified locations along the retinal section. This method is both precise and accurate and permits comparisons across sections and across animals.     

Availability of 11-cis retinal and opsins without chromophore as revealed by small bleaches of rhodopsin in excised albino mouse eyes

Small bleaches were used to study the rhodopsin regeneration process. At bleaches from 5.2% to 24.7%, the rhodopsin regenerations were consistent with a one-for-one recovery of bleached molecules. At response saturation rod photoreceptors exhibit a bleach level of only 5%. Major increases in rhodopsin regeneration were observed at bleach levels between 1.3% and 5.2%. The rhodopsin regenerations exhibited a linear relationship that was 4-times the bleach (dark adaptations of 0.75 and 1.5 h). The data show that the bleach initiates the availability, and possibly production, of 11-cis retinal in amounts that are 4-times the number of bleached molecules within the functional range of the rod photoreceptors. Rhodopsin regeneration also requires the presence of opsins without chromophore. Regenerations beyond the bleach indicate the presence of such opsins prior to the bleach. The opsin amounts were 8.1%, 8.6%, 3.1% and 0% of the total visual pigment at dark adaptation times of 0.75, 1.5, 24 and 48 h, respectively. Those opsins, as well as the ones produced by the bleach, may be regenerated to rhodopsin following a small bleach or with additional time in the dark.     

Towards a thermodynamic definition of efficacy in partial agonism: The thermodynamics of efficacy and ligand proton transfer in a G protein-coupled receptor of the rhodopsin class

The thermodynamic binding profiles of agonist and antagonist complexes of the 4-hydroxypropanolamine partial agonist, prenalterol, on the chronotropic adrenergic response in guinea-pig right atria were determined over a 15 °C temperature range. The tissue response was compared with data on the ethanolamine agonist, isoprenaline, given by binding studies in a number of rat tissues. Utilising the residue conservatism surrounding the known active conformers bound to either of two aspartate residues (α-helices II, III) in both receptors (β₁, β₂) and species (guinea-pig, rat and human), no significant deformation in the extended side chain could be found in prenalterol's agonist binding compared to isoprenaline. Antagonist binding gave a highly favourable entropy contribution at 30.0 °C of −4.7 ± 1.2 kcal/mol. The enthalpy change between bound agonist and antagonist complexes, a function of the efficacy alone, was −6.4 ± 1.1 kcal/mol, coincident with the calculated intrinsic preference of a primary/secondary amine-aspartate interaction for a neutral hydrogen-bonded form over its ion pair state, giving values of 6.3-6.6 kcal/mol with calculations of good quality, a figure expected to be close to that shown within a hydrophobic environment. Delivery of a proton to a conserved aspartate anion (α-helix II) becomes the critical determinant for agonist action with resultant proton transfer stabilisation dominating the enthalpy change. A proposed monocation-driven ligand proton pumping mechanism within the ternary complex is consistent with the data, delivery between two acid groups being created by the movement of the cation and the counter-movement of the ligand protonated amine moving from Asp 138 (α-helix III) to Asp 104 (α-helix II).     

常染色体显性视网膜色素变性家系基因定位的研究与视紫红质基因突变的检测分析

对一常染色体显性视网膜色素变性 (autosomaldominantretinitispigmentosa ,ADRP)大家系进行基因定位 ,并检测该家系12名患者的视紫红质基因是否存在突变。方法 :采用多个已知位点的遗传标记对该ADRP家系进行连锁分析 ,确定致病基因的大致染色体位置 ;在所定位的染色体区域将RHO基因作为侯选基因进行直接测序检测突变。结果 :连锁分析结果发现遗传标记D3S12 92 ,当θ =0 1时有最大Lod值 =2 732 85 2 ,因此考虑该家系致病基因位于D3S12 92附近。直接测序结果发现该家系中大部分患者在RHO基因的第 3外显子序列 ,第 182密码子的第 2个碱基发生G→A置换突变 ,导致甘氨酸 (Gly)变为天冬氨酸 (Asp) ,命名为Gly -182 -Asp突变 ,而在 2例患者中则未发现突变 ;同时 ,在该家系正常成员以及正常对照者中均未发现此突变。结论 :ADRP存在分子水平的遗传异质性 ,某些ADRP是由于RHO基因突变所致。但是由于本研究所涉及的ADRP家系中尚有2名患者未找到RHO基因突变 ,故不能将Gly -182 -Asp突变认为是该家系的致病原因。在D3S12 92与RHO基因之间可能存在新的基因 ,还需进一步研究证明。     

常染色体显性遗传视网膜色素变性患者视紫红质基因突变的研究

目的探讨常染色体显性遗传型视网膜色素变性(autosomal dominant retinitis pigmentosa,ADRP)患者视紫红质(rhodopsin,RHO)基因是否存在突变。方法应用聚合酶链反应(polymerase chain reaction,PCR)扩增RHO第1、5外显子基因片段，以限制性核酸内切酶酶切消化技术检测38个ADRP家系的57名患者和60名正常对照者RHO第58、347密码子的突变。结果1个ADRP家系的4名患者第58密码子发生点突变，另2个ADRP家系的6名患者第347密码子也出现点突变，而对照者未发现上述两种突变。结论ADRP存在分子水平的遗传异质性，某些ADRP是由于RHO基因突变所致。(中华眼底病杂志,1998,14:108-110)     

A leucine to arginine amino acid substitution at codon 46 of rhodopsin is responsible for a severe form of autosomal dominant retinitis pigmentosa

To evaluate the extent to which rhodopsin mutations are involved in autosomal dominant forms of retinitis pigmentosa (adRP) we collected DNAs from patients with adRP and screened the rhodopsin coding sequence with single-strand conformational polymorphism (SSCP) analysis and DNA sequencing. This screening revealed a thymidine to guanine transversion at nucleotide 431 (nucleotide sequence numbers as per Genebank) in affected members of one family (RFS04). The nucleotide substitution leads to a missense mutation at the 46th amino acid of rhodopsin. The mutation occurs at an amino acid conserved in mammals and changes the hydrophobic nature of the protein at a transmembrane-spanning region. The mutation causes the substitution of a non-polar hydrophobic amino acid, leucine, for the basic amino acid arginine (Leu46Arg). This nucleotide substitution is unique to the family studied and occurs in the affected individuals in the family. Full-field electroretinograms (ERGs) in four affected members of the family showed nondetectable rod responses at an early age, with markedly reduced cone responses, and a faster than average rate of progression of the phenotype as measured by yearly ERGs. © 1993 Wiley-Liss, Inc.     

The presence of a porphyropsin-based visual pigment in the juvenile lemon shark (negaprion brevirostris)

The visual pigment from the juvenile lemon shark has been extracted and is a homogeneous vitamin A₂-based porphyropsin with maximum absorption at 522 nm. This is the first report of a porphyropsin visual pigment extracted from the retina of an elasmobranch. In contrast, the visual pigment from the adult lemon shark yields a homogeneous vitamin A₁-based rhodopsin with maximum absorption at 501 nm. We conclude that the porphyropsin of the juvenile lemon shark changes over to a rhodopsin as the animal matures.