Paralogous origin of the rhodopsinlike opsin genes in lizards

Rhodopsinlike opsins constitute a distinct phylogenetic group (Yokoyama 1994, Mol. Biol. Evol. 11:32–39). This RH2 group includes the green-sensitive opsins in chicken and goldfish and the blue-sensitive opsin in a nocturnal lizard gecko. In the present study, we isolated and sequenced the genomic DNA clones for the RH2 opsin gene, rh2 _Ac , of the diurnal lizard Anolis carolinensis. This single-copy gene spans 18.3 kb from start to stop codons, making it the longest opsin gene known in vertebrates. Phylogenetic analysis strongly suggests that rh2 _Ac is more closely related to the chicken green opsin gene than to the gecko blue opsin gene. This gene tree differs from the organismal tree, where the two lizard species should be most closely related, implying that rh2 _Ac and the gecko blue-sensitive opsin genes have been derived from duplicate ancestral genes.Correspondence to: S. Yukiyama     

Differential Localization of the γ3 and γ12 Subunits of G Proteins in the Mammalian Brain

Abstract: The localization of two forms of the γ subunit of G proteins, γ₃ and γ₁₂, was examined in the mammalian brain. Concentrations of these two γ subunits increased markedly, as did those of glial fibrillary acidic protein, during postnatal development in the rat cerebral cortex. In aged human brains, by contrast, the concentration of γ₃ tended to decrease with age, whereas that of γ₁₂ in the temporal cortex increased slightly. An immunohistochemical study of human brains revealed that γ₃ was abundant in the neuropil, whereas γ₁₂ was localized in glial cells. In the hippocampal formation of aged human brains, levels of γ₁₂-positive cells, as well as levels of glial fibrillary acidic protein- and vimentin-positive astrocytes, increased, in particular in the CA1 subfield and the prosubiculum, in which there was a decrease in the number of pyramidal cells. The appearance of γ₁₂-positive cells associated with the loss of pyramidal cells was also observed in the hippocampus of rats that had been treated with kainic acid. These results indicate that γ₁₂ is strongly expressed in reactive astrocytes. In a study of cultured neural cells, we found that γ₁₂ was predominant in glioma cells, such as C6 and GA-1 cells, in contrast with the specific localization of γ₃ in PC12 pheochromocytoma cells, which are neuron-like cells. Taken together, the results indicate that γ₃ and γ₁₂ are selectively expressed in neuronal and glial cells, respectively, and that concentrations of γ₃ and γ₁₂ in the brain are related to the numbers and/or extent of maturation of these cells.     

Prostaglandin D2 lowers nuclear DNA polymerase activity in cultured mastocytoma cells

Prostaglandin D₂ strongly inhibited growth of cultured mastocytoma P-815, 2-E-6 cells, which were established and cloned from mouse mast tumor cells. The inhibition was dose-dependent (IC₅₀ = 2.09 × 10⁻⁵ M). Prostaglandin D₂ also inhibited the DNA synthesizing activity of the cells dose-dependently. We next measured the activities of endogenous DNA polymerases extracted from untreated and prostaglandin D₂-treated cells. Prostaglandin D₂ pretreatment reduced DNA polymerase α activity by 52%. The sedimentation coefficients of the enzymes from untreated and prostaglandin D₂-treated cells were the same suggesting there was no gross change in the size of the enzyme. Prostaglandin D₂ pretreatment of the cells reduced endogenous DNA polymerase β activity to 68% of the control value; the sedimentation coefficients of the enzymes from treated and untreated cells were both 3.5 S. Interestingly, prostaglandin D₂ had no direct inhibitory effect on the activity of either DNA polymerase α or β. Our results indicate that the activities of DNA polymerase α and β are lower in prostaglandin D₂-treated mastocytoma cells. This finding account for the lower level of DNA synthesis in these cells.     

Growth mechanism of myelin figures of phosphatidylcholine

The initial growth process of myelin figures, rod-like lyotropic liquid-crystalline structures, formed by phosphatidylcholine in water, ethylene glycol or glycerin, is suggested to be diffusion-limited with an apparent diffusion coefficient D of approx. 10(-6) cm2/s. D can be expressed by the sum of two processes. One is considered to describe the diffusion of an aggregate of phosphatidylcholine molecules and the other mainly to describe a lateral diffusion in the bilayer membranes which constitute myelin figures.     

Presence of adrenocorticotropin-potentiating factors in porcine thyroid glands

An extract of porcine thyroid gland in 0.1 N acetic acid exerted dose-dependent potentiation of ACTH-induced corticosterone production in isolated rat adrenal cells. The extract by itself manifested no steroidogenic activity. Upon gel-filtration of the extract, potentiating activities were demonstrated in three main peaks with molecular weights of about 10,000, 5,000 and 2,000. These findings indicate the presence of heterogeneous forms of ACTH-potentiating factors in the thyroid. Significant enhancement of ACTH-induced steroidogenesis was readily apparent with three gel-filtration fractions at a lower concentration of ACTH (4.75 pM). At this concentration, dose-dependent potentiation was observed with these three fractions. Enhanced corticosterone production responses by cells preincubated with the thyroid extract were observed and the results indicated the existence of potentiating mechanisms other than inhibition of ACTH proteolysis. The lack of T4, T3 and thyroglobulin in this activity suggests that the activity resides in other constituents of the thyroid.     

Relationship between enhancement of morphine analgesia and inhibition of enkephalinase by 2S, 3R 3-amino-2-hydroxy-4-phenylbutanoic acid derivatives

The effects of nineteen AHPA* derivatives were examined on morphine analgesia by tail-flick test in rats and on enkephalinase inhibition which was based on the formation of tyrosyl-glycyl-glycine from met-enkephalin. The correlation between the enhancement of morphine analgesia in vivo and enkephalinase inhibition in vitro was analyzed. The different analogs varied considerably in the degree of enhancement of morphine analgesia and inhibition of enkephalinase. A close relationship between enkephalinase inhibition expressed by IC50 in vitro and enhancement of morphine analgesia in vivo was observed in thirteen out of nineteen AHPA derivatives examined. One of other six AHPA derivatives which showed weak effectiveness in potentiating on morphine analgesia but was highly potent as an enkephalinase inhibitor, caused potent analgesic action when it was applied intracisternally indicating poor penetration of the blood brain barrier. The possibility was discussed that some of other compounds excluded from the linear relationship might act on other enkephalin degrading enzymes such as aminopeptidase.     

Duality of alpha-subunit of canine kidney sodium- and potassium-activated adenosinetriphophatase in electrophoresis

Sodium- and potassium-activated adenosinetriphosphatase (Na+, K+-ATPase) purified from dog kidney outer medulla was examined by polyacrylamide gel electrophoresis and by photoaffinity labeling with N-(ouabain)-N'-(2-nitro-4-azidophenyl)-ethylenediamine (NAP-ouabain). The large subunit band (alpha-band) split into two bands on the gel after the enzyme was heat-treated in the presence of 1% sodium dodecylsulfate (SDS). Of the two bands (alpha I and alpha II), alpha I had the same electrophoretic mobility as the original band, while alpha II moved slightly faster. Total conversion into alpha II was not observed, about half of the original remaining as alpha I. Below 60 degree C, heat treatment did not produce alpha II. Phenylmethylsulfonyl fluoride did not prevent the appearance of alpha II. Both alpha I and alpha II were labeled with [3H]NAP-ouabain. Nonspecific incorporation of [3H]NAP-ouabain also occurred irrespective of illumination, but it was removed either by diffusion during staining and destaining of the gel or by treatment of the enzyme with trichloroacetic acid. It is tentatively concluded that the splitting of the band reflects some intrinsic differences in situ of the alpha-subunit of dog kidney membrane Na+,K+-ATPase.     

Photochemical reaction of 9-cis-retro-γ-rhodopsin at low temperatures

9-cis-Retro-γ;rhodopsin (λ_max = 420 nm) was prepared from 9-cis-retro-γ-retinal and cattle opsin. After cooling to liquid nitrogen temperature (77 K), the pigment was irradiated with light at 380 nm. The spectrum shifted to the longer wavelengths, owing to formation of a batho product. This fact indicates that the conjugated double bond system from C-5 to C-8 of the chromophoric retinal in rhodopsin was not necessary for formation of bathorhodopsin. Reirradiation of the batho product with light at wavelengths longer than 520 nm yielded a mixture composed of presumably 9- or 11-cis forms of retro-γ-rhodopsin. These three isomers are interconvertible by light at liquid nitrogen temperature. Thus the retro-γ-rhodopsin system is similar in photochemical reaction at 77 K to cattle rhodopsin system. Each system has its own batho product. Based on these results, it was infered that the formation of bathorhodopsin is due to photoisomerization of the chromophoric retinal of rhodopsin and is not due to translocation of a proton on the ring or on the side chain from C-6 to C-8 of the chromophoric retinal to the Schiff-base nitrogen.