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PubMed1998

Functional analysis of the promoters of the human red and green visual pigment genes.

PURPOSE: To delineate cis-acting DNA elements involved in the expression of the human red and green visual pigment genes and to correlate these with transcription factor binding sites. METHODS: Assays of promoter activity were accomplished by transient transfection into WERI cells. Nested deletion and block mutagenesis were undertaken to delineate critical elements. Transcription factor binding sites were determined by DNase I footprinting and electrophoretic mobility shift (EMSA) analyses. RESULTS: The human retinoblastoma cell line WERI, but not Y-79, was found to express the red and green pigment genes. Transfection assays in WERI cells revealed that the proximal region of the red pigment gene promoter had positive (-130 to -113 and -96 to -23) and negative (-190 to -130 and - 113 to -96) regulatory elements. The green pigment gene promoter was found to be 2 to 4 times more active than was that of the red pigment. This difference in activity was attributable mainly to a T to C substitution at position -3. DNase I protection and EMSA studies demonstrated the binding of several ubiquitous and WERI-enriched proteins to DNA sequences between - 130 and the TATA box. The locus control region (LCR) did not have any enhancer activity in transient transfection. CONCLUSIONS: The WERI cell line is a good model system for the analysis of gene expression of the human cone visual pigment genes. The expression of these genes in a cell-specific fashion seems to be controlled mainly by positive-acting elements in the region between - 130 and the TATA box. The higher activity of the green pigment gene promoter could have evolved to compensate for its longer distance from the activating LCR than that of the red pigment gene promoter (approximately 34 versus 3.5 kb). Although the LCR does not enhance transcription in transient transfection, it binds factors that also recognize the proximal promoter region. These interactions may be important for the establishment of a transcriptionally active domain in a chromatin context.

Base Sequence↗
PubMed1998

Foveal cone photopigment distribution: small alterations associated with macular pigment distribution.

PURPOSE: To map the photopigment distribution of central foveal cones in healthy adult subjects before potential onset of age-related macular degeneration. To compare alterations in cone photopigment distribution to those of macular pigment and examine those loci for subretinal changes. METHODS: Eleven healthy subjects (age range, 31-59 years) underwent reflectometry with a scanning laser ophthalmoscope. The difference in cone photopigment density in the fovea was mapped for the long-wavelength- and middle-wavelength-sensitive cones, using 594-nm light. Macular pigment was mapped with 488-nm and 514-nm light. Subretinal changes were investigated with infrared imaging (830-860 nm). RESULTS: Most subjects had small alterations in the regularity of their foveal cone photopigment distribution. Alterations were spatially related to macular pigment alterations but not to the presence of subretinal defects. Subjects were classified into three groups according to the type of alterations in the regularity of pigment distributions: central peak of photopigment and macular pigment, small foveal alterations, and broad distribution with missing central peak of photopigment or macular pigment. The resultant groups differed significantly in age, 43, 46, and 59 years, for groups 1, 2, and 3, respectively (P < 0.05). CONCLUSIONS: Small alterations in the distributions of foveal cone photopigment or macular pigment were found that varied among the subjects. Larger alterations in older subjects may indicate changes in foveal architecture with age, including potential vulnerability of central cones before the onset of clinically significant changes in the retinal pigment epithelium.

Adult↗
PubMed1998

Morphological change and enhanced pigment production of monascus when cocultured with saccharomyces cerevisiae or aspergillus oryzae

When a Monascus isolate, a producer of Monascus pigments, was cocultured with either Saccharomyces cerevisiae or Aspergillus oryzae in a solid sucrose medium, there were significant morphological changes in Monascus culture. Cocultures exhibited cell mass increases of 2 times and pigment yield increases of 30 to 40 times compared to monocultures of Monascus. However, enhanced cell growth, an increase in pigment production, and morphological change did not occur in coculture with Bacillus cereus. Saccharomyces cerevisiae was more effective at enhancing pigment production than Asp. oryzae. Enhanced cell growth and increased pigment production occurred only in conjunction with morphological changes. Culture filtrates of S. cerevisiae were also effective in inducing morphology change in Monascus, similar to culture broths of S. cerevisiae. The hydrolytic enzymes produced by S. cerevisiae, such as amylase, and chitinase, are thought to be the effectors. The commercial enzymes alpha-amylase and protease from Asp. oryzae both caused a morphological change in Monascus and were effective in enhancing pigment production. However, lysozyme, alpha-amylase and protease from Bacillus species, protease from Staphylococcus, and chitinase from Streptomyces were not effective. The hydrolytic enzymes which cause a morphological change of Monascus culture and enhancement of pigment production are thought to be capable of degrading Monascus cell walls. An approximate 10-fold increase in pigment production was observed in liquid cocultures with S. cerevisiae. Copyright 1998 John Wiley & Sons, Inc.

Journal Article↗
PubMed1996

Comparison of post-junctional alpha-adrenoceptors in iris dilator muscle of humans, and albino and pigmented rabbits.

The relative potency of alpha-adrenoceptor agonists and the dissociation constants of competitive antagonists were studied to characterize the post-junctional alpha-adrenoceptor of the human iris dilator muscle. The data obtained from human iris dilator tissue was compared to that from rabbit. The iris dilator muscle was mounted in an organ bath and tension changes were recorded. (-)-Norepinephrine, (-)-phenylephrine (PE), oxymetazoline and p-aminoclonidine caused contractile responses in albino rabbit, pigmented rabbit and human iris dilator muscle in a concentration-dependent manner. The imidazoline molecules were partial agonists. In rabbit iris dilator, desensitization occurred to repeated oxymetazoline application at an interval of 1 h but recovery to the agonist activity was complete in about 3 h. Exposure to cocaine (10 mumol/l), hydrocortisone (100 mumol/l) and U-0521, a catechol-O-methyltransferase inhibitor (100 mumol/l), significantly potentiated the response to norepinephrine by 92-, 32- and 7 fold in iris dilator tissue of albino rabbit, pigmented rabbit and human, respectively. After block of "uptake1" and "uptake2", the EC50 values of norepinephrine in the albino rabbit, pigmented rabbit and human iris dilator did not differ and ranged from 99 to 195 nmol/l. Small but significant potentiation by uptake blockers was also observed in the responses to PE in the albino rabbit or pigmented rabbit iris dilator. The average maximum tension induced by 100 mumol/l PE was 96 +/- 11 mg (n = 10), 197 +/- 11 mg (n = 11), 45 +/- 5 mg (n = 27) in albino rabbit, pigmented rabbit and human iris dilator, respectively. In human iris dilator, the responses to PE were competitively antagonized by prazosin, 5-methylurapidil and phentolamine with apparent pKB values of 7.3, 6.6 and 7.5, respectively. The pKB values of the prazosin-PE interaction in iris dilator of albino and pigmented rabbit were 8.6 and 6.4, respectively. These results suggest that the post-junctional alpha-adrenoceptors in iris dilator may be similar to that in pigmented rabbit iris. The alpha-adrenoceptor of the human or pigmented rabbit iris dilator may be characterized as alpha 1L-adrenoceptor subtype. The alpha-adrenoceptor of albino rabbit iris dilator appears to be a high affinity subtype. Furthermore, albino rabbit may not be the best strain for the drug research which is relevant to human ocular therapeutics.

Adult↗
PubMed2000

Six opsins from the butterfly Papilio glaucus: molecular phylogenetic evidence for paralogous origins of red-sensitive visual pigments in insects.

It has been hypothesized that the UV-, blue-, and green-sensitive visual pigments of insects were present in the common ancestor of crustaceans and insects, whereas red-sensitive visual pigments evolved later as a result of convergent evolution. This hypothesis is examined with respect to the placement of six opsins from the swallowtail butterfly Papilio glaucus (PglRh1-6) in relationship to 46 other insect, crustacean, and chelicerate opsin sequences. All basal relationships established with maximum parsimony analysis except two are present in the distance and maximum likelihood analyses. In all analyses, the six P. glaucus opsins fall into three well-supported clades, comprised, respectively, of ultraviolet (UV), blue, and long-wavelength (LW) pigments, which appear to predate the radiation of the insects. Lepidopteran green- and red-sensitive visual pigments form a monophyletic clade, which lends support to the hypothesis from comparative physiological studies that red-sensitive visual pigments in insects have paralogous origins. Polymorphic amino acid sites (180, 197, 277, 285, 308), which are essential for generating the spectral diversity among the vertebrate red- and green-sensitive pigments are notably invariant in the Papilio red- and green-sensitive pigments. Other major tuning sites must be sought to explain the spectral diversification among these and other insect visual pigments.

5' Untranslated Regions↗
PubMed1980

The control of pigment migration in isolated erythrophores of Holocentrus ascensionis (Osbeck). I. Energy requirements.

Erythrophores isolated from the scales of the marine teleost, Holocentrus ascensionis (Osbeck), are capable of rapidly aggregating or dispersing numberous red pigment granules within their cytoplasm by translocating them along radial paths delineated by bundles of radially oriented microtubules. Pigment translocation is accompanied by transformations in the morphology of the cytoplasmic matrix, or microtrabecular lattice (MTL), in which the pigment granules are suspended. It appears that the MTL as a whole contracts toward the cell center during aggregation, carrying the pigment granules inward along with it, and is restructured during dispersion, using the radial microtubules as guides. We examined the energy requirements of pigment migration and the accompanying MTL transformations. Cellular ATP was depleted using the specific metabolic inhibitors 2,4 dinitrophenol, NaCN and oligomycin. All three of these drugs, which inhibit oxidative phosphorylation by different mechanisms, prevent both pigment dispersion and MTL transformation to dispersed morphology, while aggregation is unaffected. Inhibitor-treated cells recover normal pigment movements and MTL morphology when inhibitor is washed out of the cells with fresh medium. Potential energy apparently is stored in the MTL by some ATP-dependent process during dispersion and is converted to kinetic energy during aggregation. The results of this study strengthen the hypothesis that the MTL, working in concert with the radial microtubules, is the vehicle for pigment translocation in the erythrophore system.

Adenosine Triphosphate↗
PubMed2003

What is the risk of developing pigmentary glaucoma from pigment dispersion syndrome?

PURPOSE: To determine the probability of converting from pigment dispersion syndrome to pigmentary glaucoma. DESIGN: Retrospective community-based study of all newly diagnosed cases of pigment dispersion syndrome or pigmentary glaucoma. METHOD: Subjects were patients newly diagnosed with pigment dispersion syndrome or pigmentary glaucoma from 1976 to 1999 in Olmsted County, Minnesota. Criteria for pigment dispersion syndrome were two of three signs: midperipheral, radial iris transillumination defects; Krukenberg spindle; heavy trabecular meshwork pigmentation. Criteria for pigmentary glaucoma were pigment dispersion syndrome and two of three findings: intraocular pressure (IOP) greater than 21 mm, optic nerve damage, or visual field loss. Kaplan-Meier survival curves were used to determine the probability of conversion to pigmentary glaucoma. RESULTS: A total of 113 patients were newly diagnosed with pigment dispersion syndrome over the 24-year period. Of these, 9 persons developed pigmentary glaucoma or elevated IOP requiring therapy. The probability of converting to pigmentary glaucoma was 10% at 5 years and 15% at 15 years. An additional 23 patients were found to have pigmentary glaucoma at their initial examination. The mean age at diagnosis of pigmentary glaucoma was 42 +/- 12 years; 78% of patients were male, whereas 58% of patients with pigmentary dispersion syndrome glaucoma were male. The most significant risk factor for conversion to pigmentary glaucoma was an IOP greater than 21 mm Hg at initial examination, whereas age, refractive error, and family history of glaucoma were not correlated with conversion. CONCLUSIONS: The risk of developing pigmentary glaucoma from pigment dispersion syndrome was 10% at 5 years and 15% at 15 years. Young, myopic men were most likely to have pigmentary glaucoma. An IOP greater than 21 mm Hg at initial examination was associated with an increased risk of conversion.

Adolescent↗
PubMed2003

Long anterior zonules and pigment dispersion.

PURPOSE: To describe pigment dispersion associated with long anterior zonules. DESIGN: Multicenter observational case series. METHODS: Fifteen patients, seven of whom were treated for glaucoma or ocular hypertension, were identified with long anterior zonules and pigment dispersion. Transmission electron microscopy was performed on one anterior capsule specimen. RESULTS: All patients had anterior zonules that inserted centrally on the lens capsule. Signs of pigment dispersion included corneal endothelial pigmentation, loss of the pupillary ruff, and variable trabecular meshwork pigmentation. Ultrasound biomicroscopy verified the lack of posterior iris insertion and concavity. There was no exfoliation material. Transmission electron microscopy showed zonular lamellae with adherent pigment granules, and no exfoliation material. CONCLUSIONS: Long anterior zonules inserted onto the central lens capsule may cause mechanical disruption of the pigment epithelium at the pupillary ruff and central iris leading to pigment dispersion.

Adult↗
PubMed2001

Familial occurrence of pigment dispersion syndrome.

BACKGROUND: Pigment dispersion syndrome affects up to 4% of the white population. It is characterized by the presence of transillumination defects, Krukenberg's spindle and dense trabecular meshwork pigmentation. Open-angle glaucoma will develop in as many as 50% of affected patients. In this study we describe the familial occurrence of pigment dispersion syndrome in six North American pedigrees and the phenotypic characteristics with respect to pigment dispersion syndrome and glaucoma. METHODS: Probands with pigment dispersion syndrome were identified in glaucoma clinics at university eye centres in Ottawa and Durham, NC. Families with two or more affected members were evaluated. All willing members in each family underwent a thorough clinical examination and were classified as affected with pigment dispersion syndrome, suspect or unaffected. The previous medical records were reviewed to obtain the past medical and ocular history, including risk factors for glaucoma. RESULTS: All six families are white. Three families show at least two generations of affected members. Of the 43 subjects examined 58% were women. All 14 affected members showed moderate to heavy trabecular meshwork pigmentation and either Krukenberg's spindle or transillumination defects. The affected members were also considerably more myopic (mean spherical equivalent for the right eye -4.72 dioptres) than the suspect group or the unaffected group (mean spherical equivalent -0.79 D and +1.19 D respectively) (p < or = 0.001), and the intraocular pressure was higher for the affected than the unaffected group (mean for the right eye 20 mm Hg vs. 16 mm Hg) (p = 0.004). Half of those affected also had open-angle glaucoma. INTERPRETATION: We have identified and phenotypically characterized six North American families with autosomal dominant pigment dispersion syndrome. Our ultimate goal is to identify the gene(s) that causes this disorder in order to clarify its molecular etiology and pathophysiology. This may give rise to a molecular classification of the disease as well as provide the foundation for genetic testing and new treatment approaches.

Adult↗
PubMed2004

Intraocular pressure spike after YAG iridotomy in patients with pigment dispersion.

BACKGROUND: The role of laser peripheral iridotomy to break a suspected reverse pupil block in the long-term control of pigment dispersion is promising, but the usefulness of this procedure has not been completely established. The author examined whether patients with pigment dispersion are at higher risk for an intraocular pressure (IOP) spike after laser peripheral iridotomy due to possible compromise of trabecular meshwork function, compared with patients undergoing prophylactic peripheral iridotomy for an occludable angle. METHODS: Data were collected prospectively on the first eye of 87 patients with occludable angles and 13 patients with pigment dispersion treated with peripheral laser iridotomy between November 1995 and October 1996 at the glaucoma service of a university-affiliated hospital in Toronto. All patients received one drop of 0.5% apraclonidine before the procedure. IOP was measured before and 1 and 24 hours after the procedure. RESULTS: There was no difference between the two groups in the distribution of right vs. left eyes, sex, race, the mean total energy required to produce a patent iridotomy, the mean number of medications used or the mean IOP before the procedure. The patients with pigment dispersion were significantly younger than those with occludable angles (mean age [and standard deviation (SD)] 40.5 [9.45] years vs. 66.4 [10.78] years) (p < 0.001). There was no difference between the two groups in mean IOP at 1 hour or at 24 hours. Twenty-nine patients (33%) in the occludable angle group and seven (54%) in the pigment dispersion group had an IOP spike greater than 2 mm Hg after the procedure (p = 0.001). Among these patients, the mean IOP (36.4 [SD 10.83] mm Hg vs. 30.3 [SD 7.04] mm Hg, p = 0.05) and the mean rise in IOP (14.0 [SD 10.63] mm Hg vs. 8.7 [SD 4.73] mm Hg, p = 0.04) were significantly higher in those with pigment dispersion than in those with occludable angles. Among the patients who used antiglaucoma medications before the procedure or had a prelaser IOP level greater than 22 mm Hg, those with pigment dispersion were more likely than those with occludable angles to have an IOP spike at 1 hour (p < or = 0.005). INTERPRETATION: Patients with pigment dispersion undergoing iridotomy to break a reverse pupil block should be carefully assessed after the procedure, as significant pressure spikes requiring treatment may occur.

Aged↗
PubMed2003

Influence of aluminum doping on titania pigment structural and dispersion properties.

The influence of aluminum concentration on the structural properties and rheological behavior of aqueous suspensions of aluminum-doped titania pigment from the chloride process was investigated. The variation in rheological properties correlates with the change in the pigment surface properties, determined from electrophoresis measurements and atomic surface concentrations. Pigment suspensions exhibited a maximum yield stress and viscosity at or near the isoelectric point (iep). The pH of the maximum yield value of the pigment suspension increases with increasing aluminum hydroxyl group density at the particle surface. For pigments with a high aluminum surface concentration, at pH values where the magnitude of the zeta potential was high, a low-viscosity, dispersed suspension was obtained. The pigment with the lowest aluminum concentration, however, retained high yield stresses over a large pH range even when the zeta potential was of considerable magnitude. Pigment particle interactions are chiefly dictated by van der Waals forces and electrostatic repulsive forces, likely to be influenced by heteroaggregation. The aggregate strength would therefore depend upon the proportion and distribution of aluminum and titanium surface groups of the heterogeneous pigment, which will influence both the Hamaker constant and the degree of heteroaggregation. Overall, very small additions to the total aluminum concentration translate to significant aluminum surface concentration disparities and subsequently to large particle interaction differences.

Aluminum↗
PubMed1992

Artificial pigments of halorhodopsin and their chloride pumping activities.

Halorhodopsin (HR), the light-driven chloride pump of Halobacterium halobium, was bleached with hydroxylamine and regenerated with all-trans-retinal under several different conditions. The largest recovery of the pigment was found with apoprotein obtained from detergent-free HR [HR(BB)]. To compare the chloride-pumping mechanism of HR with that of bacteriorhodopsin (BR; the light-driven proton pump of the same bacteria), HR pigment analogues were reconstituted with the bleached HR (BB) and retinal analogues. The corresponding BR pigment analogues have previously been shown to have little or no proton-pumping activity, except for retinal2 (3,4-dehydroretinal). Pigment analogues with 13-demethylretinal or retinal2 showed an "opsin shift" similar to that of the all-trans-retinal pigment of both HR and BR. Opsin shifts of the pigments of 9-12-phenylretinal and 3,7-dimethyl-2,4,6,8-decatetraenal and haloopsin are slightly different from those of the corresponding BR pigment analogues, presumably reflecting differences of the chromophoric structures in HR and BR. In addition to the spectral properties, the effect of chloride ion on deprotonation of the Schiff base was measured. These pigment analogues showed the "chloride effect" (a shift of the pK value for deprotonation of the Schiff base), but a smaller one than that seen in HR. For a measurement of the chloride-pumping activity, each retinal analogue was added to a culture of L07 cells (BOP-, HOP+, Ret-), and the activity was measured with the cell suspension. Only cultures with retinal or retinal2 showed chloride-pumping activity, as is true for proton pumping by BR. This suggests that a similar retinal-protein interaction is necessary for both ion pumps.

Bacteriorhodopsins↗
PubMed2004

A novel amino acid substitution is responsible for spectral tuning in a rodent violet-sensitive visual pigment.

Cone short-wave (SWS1) visual pigments can be divided into two categories that correlate with spectral sensitivity, violet sensitive above 390 nm and ultraviolet sensitive below that wavelength. The evolution and mechanism of spectral tuning of SWS1 opsins are proving more complex than those of other opsin classes. Violet-sensitive pigments probably evolved from an ancestral ultraviolet-sensitive opsin, although in birds ultraviolet sensitivity has re-evolved from violet-sensitive pigments. In certain mammals, a single substitution involving the gain of a polar residue can switch sensitivity from ultraviolet to violet sensitivity, but where such a change is not involved, several substitutions may be required to effect the switch. The guinea pig, Cavia porcellus, is a hystricognathous rodent, a distinct suborder from the Sciurognathi, such as rats and mice. It has been shown by microspectrophotometry to have two cone visual pigments at 530 and 400 nm. We have ascertained the sequence of the short-wave pigment and confirmed its violet sensitivity by expression and reconstitution of the pigment in vitro. Moreover, we have shown by site-directed mutagenesis that a single residue is responsible for wavelength tuning of spectral sensitivity, a Val86Phe causing a 60 nm short-wave shift into the ultraviolet and a Val86Tyr substitution shifting the pigment 8 nm long wave. The convergent evolution of this mammalian VS pigment provides insight into the mechanism of tuning between the violet and UV.

Amino Acid Sequence↗
PubMed1998

Mechanism of spectral tuning in the dolphin visual pigments.

The absorption maxima of both rod and cone visual pigments of the bottlenose dolphin (Tursiops truncatus) are blue-shifted relative to those of terrestrial mammals. A comparison of the sequence of the dolphin rod photopigment gene with that of the bovine rod suggests that, fo the 28 nonidentical amino acids, three amino acid substitutions at positions 83, 292, and 299 in the dolphin rod pigment are responsible for the 10 nm blue shift in absorption maxima. A similar comparison of the dolphin long-wavelength sensitive (LWS) cone photopigment gene with those of the human LWS cones suggests that a single substitution at position 292 (using the convention of rhodopsin numbering) in the dolphin LWS cone pigment results in a blue shift in absorption maxima. A mutagenesis study reveals that the combination of the three dolphin specific substitutions in the bovine rod pigment (83D to 83N, 292A to 292S, and 299A to 299S) causes a blue shift from the wild-type lambdamax of 499 nm to 389 nm. The single substitution in the dolphin LWS cone pigment (292S to 292A) causes a red shift from the wild-type lambdamax of 524 nm to 552 nm. The interactions of the three amino acids identified in the rod pigment with the chromophore may be a general mechanism for blue shifting in rod visual pigments. Furthermore, the single substitution in the dolphin LWS opsin gene is a novel mechanism of wavelength modulation in mammalian LWS pigments.

Animals↗
PubMed2006

Solid state fluorescence of Pigment Yellow 101 and derivatives: a conserved property of the individual molecules.

The optical properties of the bisazomethine pigments Pigment Yellow 101 (P.Y.101) and three derivatives are investigated employing density functional theory (DFT) and time-dependent DFT (TDDFT). P.Y.101 and one of its derivatives exhibit unusual solid state fluorescence, although both possess OH groups and the latter pigment has particularly small intermolecular distances in its crystal, which are both properties that contradict common empirical rules for fluorescent pigments. Here it is shown that the OH groups are indeed essential for molecular fluorescence of the pigments due to the necessary formation of an intramolecular hydrogen bond with the lone pairs of the bisazomethine nitrogens. Furthermore, the quenching mechanism of molecular fluorescence in the non-fluorescent derivatives is analyzed in detail and a CNN bending motion of the central bisazomethine substructure is identified to be the relevant reaction coordinate along which efficient fluorescence quenching occurs in the individual molecule as well as in the crystal. Electron transfer quenching, which usually is expected to be an important quenching mechanism in aggregated media (here the crystals), is ruled out for the studied bisazomethine pigments. The solid state fluorescence properties of the pigments can finally be understood as conserved molecular properties of the individual pigment molecules.

Journal Article↗
PubMed1981

Metabolic stimulation by light in a pigmented freshwater invertebrate.

The pigmentation of terrestrial invertebrates has been shown to function in the optimization of solar warming, yet pigmented aquatic invertebrates have never been examined for the ability to utilize solar illumination in a similar manner. The degree of carotenoid pigmentation in calanoid copepods is easily quantified, and comparisons between populations suggest that variation in copepod pigmentation is related to variation in water temperature, with the most darkly pigmented copepods occurring in the coldest lakes. Darkly pigmented copepods show significantly higher metabolic rates in the light than in the dark, whereas lightly pigmented copepods do not. The metabolic experiments provide evidence that dark coloration is adaptive to cold-water copepods because it facilitates significant internal warming of tissues by sunlight. This hypothesis may be used to explain the pigmentation of many other cold-water invertebrates.

Journal Article↗
PubMed2001

Lipid peroxidation in porcine irises: dependence on pigmentation.

PURPOSE: Pigmentation of the iris is caused by varying amounts of melanin pigment granula in a constant number of melanocytes in the superficial stroma. Melanin has been shown to act as antioxidant. We have now investigated lipid peroxidation in dependence on stromal pigmentation in isolated porcine irises. METHODS: The same number of lightly and heavily pigmented porcine irises (visual selection) were homogenized (1 : 20 w/v) in buffer (50 mmol/l phosphate buffer and 4 mmol/l sodium azide). 500 microl homogenate were incubated at 37 degrees C in duplicate for 5, 10, 20 and 40 min in absence and presence of Fe2+ as inducer of lipid peroxidation. The amount of lipid peroxidation was assayed by the thiobarbituric acid (TBA) test. The results are expressed as nmol of TBA reactive material (TBAR) produced/mg protein. Fe2+ concentration of the supernatant was determined spectrophotometrically with 1,10 orthophenanthroline. Concentrations of D-glucose and D -fructose in iris tissue homogenates were determined spectrophotometrically by enzymatic bioanalysis. RESULTS: 70, 180 and 360 micromol/l Fe2+ induced lipid peroxidation. A plateau region was reached after 20 min. The amount of lipid peroxidation differed in dependence on stromal pigmentation in porcine irises. The effect was most significant at 180 micromol/l Fe2+, which induced 1.373 +/- 0.138 nmol TBAR/mg protein in lightly compared to 0.491 +/- 0.125 nmol TBAR/mg protein in heavily pigmented irises after 10 min incubation (p < 0.0001, n = 4). Similar effects (factor 2-3) were also measured after 20 and 40 min incubation. On the other hand, the content of Fe2+ in the supernatant was the same within error. Sugar concentrations (D-glucose and D-fructose) did not differ significantly for the two differently pigmented iris tissues. CONCLUSIONS: There is a stronger induction of lipid peroxidation in lightly compared to heavily pigmented porcine irises. This effect may be related to the difference in stromal melanin content and its antioxidant activity.

Animals↗
PubMed2005

Chronobiological analysis and mass spectrometric characterization of pigment-dispersing factor in the cockroach Leucophaea maderae.

Pigment-dispersing factor (PDF) is a neuropeptide that plays a prominent role in the circadian clock of several insects. The cockroach Leucophaea maderae was the first animal where the site of a biological clock could be located, and still is a focal point of circadian research. Although detailed studies on the action of pigment-dispersing factor and the distribution of PDF-like immunoreactivity in the L. maderae brain exist, a native pigment-dispersing factor of this species has not been characterized so far. The authentic Lem-PDF was isolated from L. maderae by a combination of high performance liquid chromatography, crab pigment-dispersion bioassay and an immunosorbent assay. Mass spectrometric characterization and the conserved sequence of pigment-dispersing factor in orthopteromorphan insects suggest that Lem-PDF has the sequence NSEXINSLLGLPKVLNDAa (where X= I or L). Lem-PDF is thus identical to either Periplaneta americana PDF or Acheta domesticus PDF. Detailed analysis of PDF-like immunofluorescence in different regions of the brain suggests that there are no drastic daily changes in the amount of pigment-dispersing factor as occur in Drosophila melanogaster, which might be explained by a lack of circadian pigment-dispersing factor release and production, or by phase differences between the pigment-dispersing factor neurons.

Amino Acid Sequence↗