Guanine and its retinal distribution in the tapetum of the bigeye tuna,thunnus obesus

The eye of the bigeye tuna (Thunnus obesus) contains a retinal tapetum composed of guanine. The total amount of the guanine in one eye of the fish (SL=120 cm) was about 88.6 mg. The mean guanine content of the tapetum was approximately 1.25 mg/cm² of the retinal surface. The highest content of guanine (2.15 mg/cm²) was observed only in the ventro-temporal part of the retina. To distinguish this area from the rest of the eye, we suggested the term ‘locus tapetalis’ for it. The visual accommodation system clearly indicated that the visual axis of the fish is upper-forward and the resulting retinal area for acute vision was suggested to be in the ventro-temporal retina. We discussed that the area centralis of the bigeye tuna may have two functions: to guarantee high visual acuity and to allow for high photo-sensitivity in dim light vision.     

Guanine-type retinal tapetum in the eye of the Pacific pomfret <Emphasis Type="Italic">Brama japonica</Emphasis> (Perciformes: Bramidae)

The eye of Brama japonica, which exhibits eyeshine, contains a retinal tapetum composed of guanine. The total amounts of guanine in the eyes of two specimens measuring 19.5 and 51.4cm in standard length were 7.4 and 70.5mg, respectively, the respective retinal surface areas being 4.9 and 39.9cm². The mean guanine content was almost identical (1.5 and 1.8mg/cm², respectively). A locus tapetalis, with guanine values exceeding 2.5mg/cm², was developed in the ventrotemporal region of the retina, where it cooccurred with the area centralis. Evidence is presented that sound functional reasons exist for both the development of the locus tapetalis and its position in the retina. A plea is made for future examination of the retinae of additional pelagic and nonpelagic species for the presence and location of a locus tapetalis.     

Guanine-type retinal tapetum and ganglion cell topography in the retina of a carangid fish, Kaiwarinus equula.

A guanine-type retinal tapetum was recorded in the eyes of a carangid fish Kaiwarinus equula (= Carangoides equula), spectrophotometric evidence of such being presented. The total amount of guanine in one eye was about 6.5 mg, the guanine density being ca. 1.3 mg cm(-2) over the retinal surface area. To examine the guanine distribution within the retina, the latter was divided into 21 regions. An area of high guanine density (more than 2.0 mg cm(-2)) was observed in the dorsal fundus of the retina, suggesting that the most sensitive vision was checked downward. Using whole-mount retinal preparations, the distribution of Nissl-stained cells within the retinal ganglion cell layer was examined. The greatest cell density area (area centralis) was observed only in the temporal retina. The visual acuity of the area centralis was 4.3 cycles deg(-1), suggesting that high resolution and binocular vision were directed frontally in this species. The eyes of a related carangid (Pseudocaranx dentex), lacking a tapetum, were also examined for comparison. The possible ecological advantage resulting from the tapetum is discussed in terms of visual threshold.     

Effects of hypoxia on aerobic metabolism and active electrosensory acquisition in the African weakly electric fish Marcusenius victoriae

Environmental hypoxia has effected numerous and well-documented anatomical, physiological and behavioural adaptations in fishes. Comparatively little is known about hypoxia's impacts on sensing because it is difficult to quantify sensory acquisition in vivo. Weakly electric fishes, however, rely heavily on an easily-measurable sensory modality—active electric sensing—whereby individuals emit and detect electric organ discharges (EODs). In this study, hypoxia tolerance of a mormyrid weakly electric fish, Marcusenius victoriae, was assessed by examining both its metabolic and EOD rates using a critical threshold (p_crit) paradigm. The routine metabolic rate was 1.42 mg O₂ h⁻¹, and the associated critical oxygen tension was 14.34 mmHg. Routine EOD rate was 5.68 Hz with an associated critical tension of 15.14 mmHg. These metabolic indicators of hypoxia tolerance measured in this study were consistent with those in previous studies on M. victoriae and other weakly electric fishes. Furthermore, our results suggest that some aerobic processes may be reduced in favour of maintaining the EOD rate under extreme hypoxia. These findings underscore the importance of the active electrosensory modality to these hypoxia-tolerant fish.     

A transient accumulation of poly(A)-containing RNA in the tapetum of Hyoscyamus niger during microsporogenesis

The distribution of poly(A)-containing RNA in the tapetal cells of Hyoscyamus niger during microsporogenesis was followed by in situ hybridization with [³H]poly(U) as a probe. Although no poly(A)-containing RNA accumulated in the premeiotic tapetum, [³H]poly(U) binding sites were detected in the tapetum as meiosis was completed in the microsporocytes. Accumulation of poly(A)-containing RNA in the tapetal cells reached a peak before the first haploid mitosis in the pollen grains. With the onset of tapetal senescence at the late uninucleate stage of the pollen grain, [³H]poly(U) binding sites gradually decreased and they completely disappeared in the tapetum at the binucleate pollen stage. The significance of the results is discussed, particularly with regard to the possible role of tapetum in the synthesis of informational macromolecules during microsporogenesis.     

NMR and FT-IR conformational studies of 8-substituted guanine nucleosides and nucleotides and their metal adducts and cancer

NMR and FT-IR Studies of the conformational changes of guanosine and guanosine-5′-monophosphate upon substitution of the H8 of guanine by a heavy, large atom, such as bromine, are presented. The conformational forms, syn, anti, C2′-endo and C3′-endo and gg, gt and tg rotamers of the above molecules are compared to those of their metal (Mg²⁺ and P^t2+) adducts, where the metal is fixed to the N7 nitrogen atom of guanine. The antitumor activity of cisplatin is discussed with relation to the conformational form and the effect of cisplatin is compared to the effects of the Mg²⁺ ion and carcinogens.     

Formation of the N2-acetyl-2,6-diaminopurine oligonucleotide impurity caused by acetyl capping

The acetyl ‘capping’ reaction routinely employed during phosphorothioate oligonucleotide synthesis has been implicated in the formation of an impurity species with a mass 41 amu greater than the expected oligonucleotide molecule. The impurity has been found to arise by conversion of a protected guanine nucleobase to N²-acetyl-2,6-diaminopurine. A two-part mechanism is proposed consisting of transamidation of the protecting group on guanine and substitution of guanine’s O⁶ atom.     

The photosensitive retinal pigments of fishes from relatively turbid coastal waters

Digitonin extracts have been prepared from the retinae of a dozen species of marine and euryhaline teleost fishes from turbid water habitats. Spectrophotometric analysis of the extracts shows that the photosensitive retinal pigments of these species have maximum absorption above 500 mµ. In nine species there are retinene₁ pigments with λ_max between 504 and 512 mµ. In the marine but euryhaline mullet, Mugil cephalus, there is a porphyropsin with λ_max 520 mµ. A mixture of rhodopsin and porphyropsin in an extract of a marine puffer, Sphoeroides annulatus, was disclosed by partial bleaching with colored light. In addition, one other species has a 508 mµ pigment, of which the nature of the chromophore was not determined. The habitats in which these fishes live are relatively turbid, with the water greenish or yellowish in color. The spectral transmission of such waters is probably maximal between 520 and 570 mµ. It is suggested that the fishes have become adapted to these conditions by small but significant shifts in spectral absorption of their retinal pigments. These pigments are decidedly more effective than rhodopsin in absorption of wavelengths above 500 mµ. This offers a possible interpretation of the confusing array of retinal pigments described from marine and euryhaline fishes.     

A transversion mutation hypothesis for chemical carcinogenesis by N2-substitution of guanine in DNA

Several carcinogenic aromatic amines and polycyclic hydrocarbons react covalently with the exocyclic amino group (N²) of guanine in DNA. In this study, space-filling molecular models of DNA containing N²-guanyl adducts of 2-acetylaminofluorene (AAF) or benzo[a]pyrene (BP) were constructed. From these models and from available physico-chemical data, it is suggested that the N² adducts may be easily converted from the normal anti to a syn conformation (base/deoxyribose). This confuguration causes minimal distortion of the DNA model with only a 2–3 Å shift in the helical axis of symmetry. Such an alteration may account for the persistence of these adducts in DNA and for the frameshift mutations induced by these carcinogens. Additionally, the syn N²-guanyl configuration places the N-7 and O⁶ atoms of the modified syn guanine in the base pairing region such that, during replication, mispairing with N-1 and N² of an opposite guanine may occur. This would then represent a carcinogen-induced transversion mutation and may lead to neoplastic transformation.     

Fine structure of the retinal epithelium and tapetum lucidum of the opossum (didelphis virginiana)

The fine structure of the retinal epithelium has been studied by electron microscopy in the opossum (Didelphis virginiana). The retinal epithelium, over most of the retina, is typical of that in other vertebrates and consists of a single layer of heavily pigmented, cuboidal cells. These cells display extensive basal (scleral) infoldings and numerous apical (vitreal) processes which enclose photoreceptor outer segments. A semicircular area of the retinal epithelium in the superior fundus is further specialized as a tapetum lucidum. The reflecting material consists of a large quantity of lipoidal spheres scattered throughout the epithelial cells. Centrally in the tapetal area very few or no melanosomes are found, indicating a non-occlusible tapetum. Peripherally in the tapetum, the epithelial cells contain both reflecting material and melanosomes. As in the non-tapetal area, the epithelial cells of the tapetum display large amounts of smooth endoplasmic reticulum and numerous mitochondria. Bruch's membrane everywhere displays the usual pentalaminate structure described for most vertebrates. The choriocapillaris is also typical, in that numerous fenestrations are present in the endothelium bordering Bruch's membrane.     

Evidence for the presence of ion pumps in the photophores of two mesopelagic fishes: Argyropelecus and maurolicus

• 1.l. Adenosine triphosphatase (ATPase) activity has been measured on homogenates of photophores from the two mesopelagic fishes Argyropelecus and Maurolicus. This activity is equivalent for both fishes when reported to the protein content as is their O₂ consumption.
• 2.2. The activity is optimal at pH 6.8–7.5. It is not specific for ATP since GTP, ITP, UTP and CTP are also hydrolyzed to a significant extent. It is also not specific for Mg²⁺, the activity being equivalent (Argyropelecus) or higher (Maurolicus) with Ca²⁺ and high also with Co²⁺ and Mn²⁺
• 3.3. Twenty to 30 per cent of the activity measured at pH 7.4 is probably due to the mitochondrial ATPase as it is shown by oligomycin and venturicidin inhibition.
• 4.4. Activities of both fishes photophores are partly inhibited by N-N'-dicyclohexylcarbodiimide (DCCD), azide, LaCl₃, vanadate, diethylstilbestrol (DES) and N-ethylmaleimide (NEM) which are all inhibitors of ionic pumps.
• 5.5. Argyropelecus activity is sensitive to ouabaïn.
• 6.6. Our results show the presence of ionic pumps in Argyropelecus and Maurolicus photophores. If there is evidence for the absence or very low activity of a H⁺ pump, it is sure that Argyropelecus at least possess a Na⁺K⁺-ATPase.
• 7.7. The significance of a high protein content in Maurolicus photophores and of a large inorganic phosphate concentration in Argyropelecus is discussed.

     

A method using 3-O-methyl-D-glucose and phloretin for the determination of intracellular water space of cells in monolayer culture.

A method is presented for determining the extent of methylation of tRNAs synthesized in mammalian and bacterial cell systems and is based upon determining the distribution of radioactivity associated with the guanine constituents of total cellular tRNA preparations previously labeled with [2-¹⁴C]guanosine and with [methyl]-³H or -¹⁴C]methionine. Whereas labeling with guanosine provides a means of assessing the extent of methylation of the [2-¹⁴C]guanine residues incorporated into tRNA, methionine labeling provides a measure of the percentage of [methyl-³H or -¹⁴C]methylated constituents that are methylated guanines. Analyses such as the above reveal that the tRNA of KB cells acquires approximately three times as many methyl groups as that of E. coli B tRNA. Coupled with the knowledge that both mammalian and bacterial tRNA preparations contain an average of 24 guanine residues per molecule, the above analyses further reveal that 7.2 and 2.4 methyl groups are incorporated into each tRNA molecule synthesized in exponentially growing KB- and E. coli B-cells, respectively. Additional information regarding the extent of formation of individual methylated constituents per tRNA molecule synthesized is presented.     

Biosynthesis of riboflavine by a purine-requiring mutant strain of Escherichia coli

1. Riboflavine biosynthesis occurs in non-proliferating cultures of a purine-requiring strain of Escherichia coli (ATCC no. 13863). 2. No significant incorporation of radioactivity from [1-¹⁴C]glycine into either C-4a and C-9a of riboflavine or into nucleic acid purines is detected under the above conditions; appreciable incorporation of label into 5-aminoimidazole-4-carboxamide occurs. However, the label of [6-¹⁴C]guanine is incorporated significantly into C-4 of riboflavine and into nucleic acid adenine and guanine; the specific radioactivity of the riboflavine is approximately twice that of either adenine or guanine of nucleic acid. 3. These results show that a purine derivative is an obligatory intermediate in riboflavine biogenesis.     

Photoreceptor Proteins Initiate Microglial Activation via Toll-like Receptor 4 in Retinal Degeneration Mediated by All-trans-retinal

Although several genetic and biochemical factors are associated with the pathogenesis of retinal degeneration, it has yet to be determined how these different impairments can cause similar degenerative phenotypes. Here, we report microglial/macrophage activation in both a Stargardt disease and age-related macular degeneration mouse model caused by delayed clearance of all-trans-retinal from the retina, and in a retinitis pigmentosa mouse model with impaired retinal pigment epithelium (RPE) phagocytosis. Mouse microglia displayed RPE cytotoxicity and increased production of inflammatory chemokines/cytokines, Ccl2, Il1b, and Tnf, after coincubation with ligands that activate innate immunity. Notably, phagocytosis of photoreceptor proteins increased the activation of microglia/macrophages and RPE cells isolated from model mice as well as wild-type mice. The mRNA levels of Tlr2 and Tlr4, which can recognize proteins as their ligands, were elevated in mice with retinal degeneration. Bone marrow-derived macrophages from Tlr4-deficient mice did not increase Ccl2 after coincubation with photoreceptor proteins. Tlr4^−/−Abca4^−/−Rdh8^−/− mice displayed milder retinal degenerative phenotypes than Abca4^−/−Rdh8^−/− mice. Additionally, inactivation of microglia/macrophages by pharmacological approaches attenuated mouse retinal degeneration. This study demonstrates an important contribution of TLR4-mediated microglial activation by endogenous photoreceptor proteins in retinal inflammation that aggravates retinal cell death. This pathway is likely to represent an underlying common pathology in degenerative retinal disorders.     

Critical Endothelial Regulation by LRP5 during Retinal Vascular Development

Vascular abnormalities in the eye are the leading cause of many forms of inherited and acquired human blindness. Loss-of-function mutations in the Wnt-binding co-receptor LRP5 leads to aberrant ocular vascularization and loss of vision in genetic disorders such as osteoporosis-pseudoglioma syndrome. The canonical Wnt-β-catenin pathway is known to regulate retinal vascular development. However, it is unclear what precise role LPR5 plays in this process. Here, we show that loss of LRP5 function in mice causes retinal hypovascularization during development as well as retinal neovascularization in adulthood with disorganized and leaky vessels. Using a highly specific Flk1-Cre^Breier line for vascular endothelial cells, together with several genetic models, we demonstrate that loss of endothelium-derived LRP5 recapitulates the retinal vascular defects in Lrp5^-/- mice. In addition, restoring LRP5 function only in endothelial cells in Lrp5^-/- mice rescues their retinal vascular abnormalities. Furthermore, we show that retinal vascularization is regulated by LRP5 in a dosage dependent manner and does not depend on LRP6. Our study provides the first direct evidence that endothelium-derived LRP5 is both necessary and sufficient to mediate its critical role in the development and maintenance of retinal vasculature.     

Cholera-toxin-dependent ADP-ribosylation of the adenylate cyclase regulatory protein in turkey erythrocyte membranes

Incubation of turkey erythrocyte membranes with cholera toxin and [³²P]NAD caused toxin-dependent incorporation of ³²P into a 42,000 M_r peptide which could be distinguished from toxin-independent ³²P incorporation into other membrane proteins. The radiolabeled 42,000 M_r peptide could be extracted from the membranes using Lubrol PX. When toxin-treated membranes were incubated with isoproterenol and GMP before detergent solubilization, the 42,000 M_r labeled peptide was adsorbed by GTP-γ-agarose which, with the same conditions, adsorbed the adenylate cyclase guanine nucleotide regulatory protein. The labeled peptide and guanine nucleotide regulatory protein activity were coeluted from the affinity matrix by guanylyl-β,γ-imidodiphosphate, GDP, and GMP. Guanosine 5′-O-(2-thiodiphosphate), an analog of GDP which blocks guanine nucleotide- and fluoride-stimulated adenylate cyclase activity, caused elution of labeled peptide which exhibited no regulatory protein activity. Our data support the view that the 42,000 M_r peptide is part of the adenylate cyclase guanine nucleotide regulatory protein. The labeled peptide allows identification of both active and inactive regulatory protein and should be useful in monitoring the purification of the regulatory protein from turkey erythrocytes.     

A highly conserved zebrafish IMPDH retinal isoform produces the majority of guanine and forms dynamic protein filaments in photoreceptor cells

Inosine monophosphate dehydrogenase (IMPDH) is a key regulatory enzyme in the de novo synthesis of the purine base guanine. Dominant mutations in human IMPDH1 cause photoreceptor degeneration for reasons that are unknown. Here, we sought to provide some foundational information on Impdh1a in the zebrafish retina. We found that in zebrafish, gene subfunctionalization due to ancestral duplication resulted in a predominant retinal variant expressed exclusively in rod and cone photoreceptors. This variant is structurally and functionally similar to the human IMPDH1 retinal variant and shares a reduced sensitivity to GTP-mediated inhibition. We also demonstrated that Impdh1a forms prominent protein filaments in vitro and in vivo in both rod and cone photoreceptor cell bodies, synapses, and to a lesser degree, in outer segments. These filaments changed length and cellular distribution throughout the day consistent with diurnal changes in both mRNA and protein levels. The loss of Impdh1a resulted in a substantial reduction of guanine levels, although cellular morphology and cGMP levels remained normal. Our findings demonstrate a significant role for IMPDH1 in photoreceptor guanine production and provide fundamental new information on the details of this protein in the zebrafish retina.     

Polarizing optics in a spider eye

Many arthropods including insects and spiders exploit skylight polarization for navigation. One of the four eye pairs of the spider Drassodes cupreus is dedicated to detect skylight polarization. These eyes are equipped with a tapetum that strongly plane-polarizes reflected light. This effectively enhances the polarization-sensitivity of the photoreceptors, improving orientation performance. With a multidisciplinary approach, we demonstrate that D. cupreus exploits reflective elements also present in non-polarizing tapetal eyes of other species such as Agelena labyrinthica. By approximately orthogonal arrangement of two multilayer reflectors consisting of reflecting guanine platelets, the tapetum uses the mechanism of polarization by reflection for polarizing reflected light.     

tRNA methylases from HeLa cells: purification and properties of an adenine-1-methylase and a guanine-N2-methylase

Two enzymes (methylases) that catalyze the transfer of methyl groups from S-adenosyl-l-methionine to tRNA (prepared from Escherichia coli) have been partially purified from extracts of HeLa cells. One catalyzes the methylation of adenine residues of the tRNA to give 1-methyladenine units and the other is responsible for the conversion of guanine residues to N²-methylguanine and N²,N²-dimethylguanine (and may be a mixture of two enzymes). Activities of these relatively unstable enzymes could be maintained by storage at ?20 °C in the presence of 50% glycerol. Substrate specificity studies have revealed that bacterial tRNA (E. coli, Bacillus subtilis) can be used as substrate, whereas tRNA of animal origin (HeLa cells, rat liver) cannot be used. Of the specific tRNA's tested, E. coli tRNA^fMet was used as substrate by both enzymes. E. coli tRNA^Tyr was used by the adenine-1-methylase but not by the guanine-N²-methylase. The adenine-1-methylase catalyzed the transfer of approximately one methyl group per mole of either tRNA^fMet or tRNA^Tyr offered as substrate; in the presence of the guanine-N²-methylase 1 mole of E. coli tRNA^fMet accepted 1 mole of methyl. Studies with the use of both enzymes established that enzymic methylation of the guanine site of E. coli tRNA^fMet did not interfere with subsequent methylation of an adenine residue and neither did prior methylation of adenine interfere with the subsequent methylation of a guanine residue. In the presence of both enzymes, approximately 2 moles of methyl groups were accepted by 1 mole of the E. coli tRNA^fMet.