Proton-pumping rhodopsin (PPR) is a simple photosystem widely distributed in nature. By binding to retinal, PPR can transfer protons from the cytoplasmic to the extracellular side of the membrane under illumination, creating a proton motive force (PMF) to synthesize ATP. The conversion of light into chemical energy by introducing rhodopsin into nonphotosynthetic engineered strains could contribute to promoting growth, increasing production and improving cell tolerance of microbial hosts. Gloeorhodopsin (GR) is a PPR from Gloeobacter violaceus PCC 7421. We expressed GR heterologously in Escherichia coli and verified its functional activity. GR could properly function as a light-driven proton pump and its absorption maximum was at 539 nm. We observed that GR was mainly located on the cell membrane and no inclusion body could be found. After increasing expression level by ribosome binding site optimization, intracellular ATP increased, suggesting that GR could supply additional energy to heterologous hosts under given conditions.
Cyanobacteria/metabolism* ; Escherichia coli/metabolism* ; Proton Pumps ; Rhodopsin/metabolism* ; Rhodopsins, Microbial/metabolism*

PURPOSE: The purpose of this study was to clarify the effects of zinc treatment and hypothermia on visual adaptation and visual sensitivity in bullfrogs (Rana catesbeiana), which are poikilothermal animals capable of adjusting quickly to environmental temperature changes. METHODS: The effects of both zinc treatment and hypothermia on visual sensitivity were studied by using electroretinogram (ERG) recording and absorption spectra scanning before and after zinc and TSQ (N-[6-methoxy-8-quinolyl]-p-toluene sulfonamide) treatment, with or without temperature changes. RESULTS: In spite of malnutrition due to hibernation, the optimal zinc concentration effect was obtained at 10-4 M (10-2 M 200 microliter ZnCl2 in 20 microliter Ringer's solution) according to ERG recording. After zinc treatment and hypothermia induction, increments of all ERG components and thresholds were taken by ERG recording. These results showed that both zinc treatment and hypothermia may increase visual sensitivity during visual adaptation. In spectral scans, the absorbance increment due to zinc treatment and hypothermia was shown over the whole spectral range (400~750 nm), and it was especially prominent at alpha-peak (about 500 nm). In addition, there was a decrease in absorption differences between dark adaptation and light adaptation after zinc treatment. Furthermore, according to the visual sensitivity decrement using TSQ as a zinc specific chelator, this visual sensitivity increase was shown to be caused by zinc. CONCLUSIONS: As the results suggest, both zinc treatment and hypothermic effects may improve visual sensitivity by promoting rhodopsin regeneration and inhibiting rhodopsin bleaching induced by light illumination. Zinc may activate the enzyme activity of retinol dehydrogenase and phosphodiesterase, while hypothermic effects may improve precursor transport, which is required for rhodopsin regeneration, by tightening membrane adhesion between retinas and retinal pigment epithelia. In addition, we believe that zinc treatment and hypothermic effects may work synergistically to accelerate visual sensitivity during visual adaptation.
Absorption ; Adaptation, Ocular ; Animals ; Dark Adaptation* ; Hibernation ; Hypothermia ; Lighting ; Malnutrition ; Membranes ; Oxidoreductases ; Rana catesbeiana ; Regeneration ; Retina* ; Retinaldehyde ; Rhodopsin ; Vertebrates* ; Vitamin A ; Zinc*

Recoverin is a member of the large family of EF-hand calcium binding proteins (Baimbridge et al., 1992), and it is thought to be involved in the regulation of phosphodiesterase in photoreceptors and in the phosphorylation of activated rhodopsin (Polans et al., 1996). Although the functional significance of recoverin in cone bipolar cells is not fully understood, the antiserum against recoverin has been widely used to identify a certain population of cone bipolar cells (Milam et al., 1993; Sasso's Pognetto et al., 1994; Euler & W sle, 1995). GABA is well known to act as major neurotransmitters in the mammalian central nervous system including retina. This study was conducted to identify the development process of recoverin-labeled cone bipolar cells, and the timing points of synaptic formation of the labeled bipolar cells and GABAergic amacrine cells in the rat retina. The results were as follows; In the adult rat retina, recoverin-labeled cone bipolar cells were subdivided into twotypes; type 2 cells with axon terminal stratified in sublamina a of the inner plexiform layer (IPL), and type 8 cells with axon terminals stratified in sublamina b of the IPL. Recoverin-labeled cone bipolar cells began to appear from postnatal day 5. The axon terminals of recoverin-labeled type 2 cone bipolar cells stratified at postnatal day 10, while those of type 8 cone bipolar cells stratified at postnatal day 13. The axon terminals of type 2 cone bipolar cells made ribbon synapses onto GABAergic amacrine cells in the IPL at postnatal day 10. These results demonstrate that recoverin-labeled type 2 cone bipolar cells differentiate earlier than recoverin-labeled type 8 cone bipolar cells, and suggest that GABAergic amacrine cells may play important roles in visual processing of recoverin-labeled type 2 cone bipolar cells by making synapse onto these cells at early stage. Synapses between type 2 cone bipolar cells and GABAergic amacrine cells are formed about the time of postnatal day 10 for visual processing.
Adult ; Amacrine Cells* ; Animals ; Calcium-Binding Proteins ; Central Nervous System ; gamma-Aminobutyric Acid ; Humans ; Neurotransmitter Agents ; Phosphorylation ; Presynaptic Terminals ; Rats* ; Recoverin* ; Retina* ; Rhodopsin ; Synapses

OBJECTIVETo detect mutation in the rhodopsin gene (RHO) in a Chinese family with autosomal dominant retinitis pigmentosa (ADRP).

METHODSA total of 25 family members from a Chinese family were investigated. All the subjects were examined clinically by direct funduscopy, perimetry and vision test. Evaluation of the proband included electroretinography (ERG). Genomic DNA was extracted using standard method. The complete coding regions of RHO were amplified by polymerase chain reaction (PCR) and the PCR products were subjected to automatic DNA sequencing.

RESULTS512 C>T (P171L), a recurrent missense mutation was detected in the proband. All 12 affected subjects in the family were heterozygous for the mutation. The affected individuals had night blindness at the age of 5-6 years. They had relatively severe impairment of visual acuity and suffered a gradual loss of peripheral visual field at the age of 20-30 years. And they went blind at the age of 40-50 years. Rod and cone ERG were not detectable in the proband.

CONCLUSIONA recurrent missense mutation, 512C>T (P171L), was detected in a Chinese family with ADRP.

Adolescent ; Adult ; Base Sequence ; China ; DNA Mutational Analysis ; Family Health ; Female ; Humans ; Male ; Middle Aged ; Mutation, Missense ; Pedigree ; Polymerase Chain Reaction ; Retinitis Pigmentosa ; genetics ; pathology ; Rhodopsin ; genetics

INTRODUCTIONRetinitis pigmentosa (RP) describes a group of inherited disorders characterised by progressive retinal dysfunction, cell loss and atrophy of retinal tissue. RP demonstrates considerable clinical and genetic heterogeneity, with wide variations in disease severity, progression, and gene involvement. We studied a large family with RP to determine the pattern of inheritance and identify the disease-causing mutation, and then to describe the phenotypic presentation of this family.

MATERIALS AND METHODSOphthalmic examination was performed on 46 family members to identify affected individuals and to characterise the disease phenotype. Family pedigree was obtained. Some family members also had fundus photographs, fluorescein angiography, and/or optical coherence tomography (OCT) analysis performed. Genetic linkage was performed using short tandem repeat (STR) polymorphic markers encompassing the known loci for autosomal dominant RP. Finally, DNA sequencing was performed to identify the mutation present in this family.

RESULTSClinical features included nyctalopia, constriction of visual fields and eventual loss of central vision. Sequence analysis revealed a G-to-T nucleotide change in the Rhodopsin gene, predicting a Gly-51-Val substitution.

CONCLUSIONSThis large multi-generation family demonstrates the phenotypic variability of a previously identified autosomal dominant mutation of the Rhodopsin gene.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Child ; Female ; Genes, Dominant ; Humans ; Male ; Middle Aged ; Mutation ; Pedigree ; Retinitis Pigmentosa ; genetics ; Rhodopsin ; genetics

G-protein coupled receptors form a large superfamily of plasma membrane proteins which serve a variety of signal transduction roles. New receptors continue to be identified. Based on sequence homology the superfamily can currently be divided into three families, the rhodopsin family which includes the vast majority of identified receptors, and the secretin. and metabotropic glutamate receptor families which share a general architecture with each other and the rhodopsin family but no obvious sequence identity. Screening for additional members of the secretin family led to the identification of the parathyroid hormone-2 (PTH2) receptor. Ligand recognition by the PTH2 receptor partially overlaps that of the PTH/parathyroid hormone-related peptide (PTHrP) receptor. This has facilitated structure-function analysis of ligands for these receptors. The physiological role of the PTH2 receptor is under investigation but its distribution suggests that it may be a neurotransmitter receptor and could participate in modulation of a number of organ systems. The relative abundance of PTH2 receptor mRNA in the brain and the inability to detect mRNA encoding PTH, its only currently identified ligand, suggest the existence of another endogenous ligand, for which evidence has recently been obtained.
Brain ; Cell Membrane ; GTP-Binding Proteins ; Humans ; Ligands ; Mass Screening ; Neurotransmitter Agents ; Parathyroid Hormone ; Receptor, Parathyroid Hormone, Type 2 ; Receptors, Metabotropic Glutamate ; Receptors, Neurotransmitter ; Rhodopsin ; RNA, Messenger ; Secretin ; Sequence Homology ; Signal Transduction

OBJECTIVETo determine the causative mutation in a 5 generation pedigree with autosomal dominant retinitis pigmentosa (ADRP).

METHODSGenomic DNA from four patients and 4 normal persons in the same pedigree suffering ADRP were extracted, and subsequently eight exons of three ADRP candidate genes were screened for mutations by a combined polymerase chain reaction-single strand conformation polymorphism and DNA sequencing techniques.

RESULTSA new point mutation in rhodopsin gene at codon 52 of exon 1 (TTC to TAC) that resulted in a substitution of Tyr to Phe was detected in the four affected family members, but not in the four control individuals from the same pedigree.

CONCLUSIONA causative mutation of rhodopsin gene was identified in a large Chinese pedigree with ADRP. The present study confirmed the molecular genetic heterogeneity of ADRP.

Adult ; Base Sequence ; DNA ; chemistry ; genetics ; DNA Mutational Analysis ; Family Health ; Female ; Genes, Dominant ; genetics ; Genetic Predisposition to Disease ; genetics ; Genetic Testing ; Humans ; Male ; Middle Aged ; Mutation, Missense ; Polymorphism, Single-Stranded Conformational ; Retinitis Pigmentosa ; diagnosis ; genetics ; Rhodopsin ; genetics

OBJECTIVETo test the frequency and pattern of rhodopsin (RHO) mutations in Chinese retinitis pigmentosa (RP) patients and to evaluate their effects in the pathogenesis of RP.

METHODSGenomic DNA was extracted from peripheral blood samples of 100 Hong Kong Chinese RP patients. Sequence variants of the entire coding exons of the RHO gene were tested using PCR, conformation sensitive gel electrophoresis and DNA sequencing.

RESULTSTotally six nucleotide changes were identified, among which three were silent mutations, two missense mutations and one deletion mutation. P347L was found in one RP proband and her three children who also had RP. P327(1 bp del) was novel and detected in a late-onset RP patient of 53 years. Her 26-year-old daughter, also carrying the identified mutation, had no RP phenotypes except for the mottled retinal pigment epithelium (RPE) revealed by fundal examination. Neither of the two mutations was detected in normal controls.

CONCLUSIONTwo patients had disease-causing mutations in the RHO gene, thus RHO mutations cause about 2.0% (95% confidence interval: 0.2%-7.0%) of all RP among Chinese in Hong Kong. A highly conserved C-terminal sequence QVS(A)PA was altered due to P347L and thereby resulting in an aberrant subcellular localization of rhodopsin. Loss of all six phosphorylatable residues at the C-terminus and the highly conserved C-terminal sequence QVS(A)PA may occur because of P327(1 bp del). To elucidate the predominant biochemical defects in such mutant, transgenic mice and transfected culture cells carrying P327(1 bp del) would be of greatest value.

Adolescent ; Adult ; Aged ; Child ; China ; DNA ; chemistry ; genetics ; DNA Mutational Analysis ; Female ; Gene Frequency ; Genetic Testing ; Humans ; Male ; Middle Aged ; Point Mutation ; Retinitis Pigmentosa ; genetics ; pathology ; prevention & control ; Rhodopsin ; genetics ; Sequence Deletion

OBJECTIVETo detect genetic mutations associated with autosomal dominant congenital stationary night blindness (ADCSNB) in a family from Henan province.

METHODSGenomic DNA was extracted from peripheral blood samples of 14 family members. Based on 3 genes reported previously, PCR primers were designed and corresponding exons containing the mutation sites were amplified with PCR. PCR products were purified and directly sequenced.

RESULTSA c.281C>T heterozygous missense mutation was detected in RHO gene in all of the patients. This mutation can cause a change of the protein structure (p.Thr94Ile). The same mutation was not detected in normal individuals from the family and 50 normal controls.

CONCLUSIONA c.281C>T mutation in RHO gene is responsible for the onset of ADCSNB in this Chinese family and results in symptoms of night blindness.

Adult ; Amino Acid Sequence ; China ; DNA Mutational Analysis ; methods ; Eye Diseases, Hereditary ; Female ; Genetic Diseases, X-Linked ; Genetic Predisposition to Disease ; Humans ; Male ; Molecular Sequence Data ; Mutation, Missense ; Myopia ; genetics ; Night Blindness ; genetics ; Rhodopsin ; genetics ; Sequence Alignment ; methods

OBJECTIVETo evaluate the prevalence of rhodopsin (RHO) mutations and the genotype-phenotype relationships in Chinese patients with autosomal dominant retinitis pigmentosa (ADRP) by conformation sensitive gel electrophoresis (CSGE) and direct DNA sequencing.

METHODSWe have screened the five coding exons and splice sites of RHO gene in 27 probands who had no relativity from Chinese ADRP families and 100 normal controls to identify disease-associated mutations, using CSGE and direct DNA sequencing. Family members of some probands with disease-associated mutations were also genotyped to determine whether the RHO mutations segregated with retinitis pigmentosa (RP) in their families.

RESULTSTwo RHO mutations, Pro347Leu and Pro327 (1-bp del), were identified separately in two families, thus the frequency of RHO mutations among this set of Chinese ADRP families is about 7.4% (2/27). Pro347Leu mutation was found in one ADRP proband as well as three her children who also had RP. She had relatively early onset at about 17 years. The only one child without this mutation had no symptom or sign of RP at age of 34. Pro327 (1-bp del) was identified in a late-onset ADRP patient, who appeared night blindness around 30 years old and in her fifties electroretinogram (ERG) has been flat in both scotopic and photopic phases. Family analysis showed that this mutation also existed in her younger daughter and her elder sister, both of them also had RP. Three other family members were genotypically and phenotypically normal. Neither of the two mutations was detected in 100 normal controls.

CONCLUSIONSThe frequency of RHO mutations in Chinese patients was lower than that in Europe and North America. The phenotype of the patients with Pro347Leu corresponded to type 1 ADRP, with severe rod degeneration and some cone preservation later, while the phenotype of the patients carrying Pro327 (1-bp del) corresponded to type 2 ADRP, with a concomitant loss of rod and cone visual function. CSGE was found to be a sensitive, simple, and practical method for the screening of a large number of samples under highly reproducible conditions, and could be utilized in routine molecular diagnostic laboratories.

Asian Continental Ancestry Group ; Base Sequence ; DNA Mutational Analysis ; DNA, Antisense ; genetics ; Electrophoresis, Polyacrylamide Gel ; methods ; Exons ; Female ; Genotype ; Humans ; Middle Aged ; Molecular Sequence Data ; Mutation, Missense ; Phenotype ; Retinitis Pigmentosa ; genetics ; Rhodopsin ; genetics