Retinal Attachment Instability Is Diversified among Mammalian Melanopsins

Melanopsins play a key role in non-visual photoreception in mammals. Their close phylogenetic relationship to the photopigments in invertebrate visual cells suggests they have evolved to acquire molecular characteristics that are more suited for their non-visual functions. Here we set out to identify such characteristics by comparing the molecular properties of mammalian melanopsin to those of invertebrate melanopsin and visual pigment. Our data show that the Schiff base linking the chromophore retinal to the protein is more susceptive to spontaneous cleavage in mammalian melanopsins. We also find this stability is highly diversified between mammalian species, being particularly unstable for human melanopsin. Through mutagenesis analyses, we find that this diversified stability is mainly due to parallel amino acid substitutions in extracellular regions. We propose that the different stability of the retinal attachment in melanopsins may contribute to functional tuning of non-visual photoreception in mammals.     

A 5-bp Insertion in Mip Causes Recessive Congenital Cataract in KFRS4/Kyo Rats

We discovered a new cataract mutation, kfrs4, in the Kyoto Fancy Rat Stock (KFRS) background. Within 1 month of birth, all kfrs4/kfrs4 homozygotes developed cataracts, with severe opacity in the nuclei of the lens. In contrast, no opacity was observed in the kfrs4/+ heterozygotes. We continued to observe these rats until they reached 1 year of age and found that cataractogenesis did not occur in kfrs4/+ rats. To define the histological defects in the lenses of kfrs4 rats, sections of the eyes of these rats were prepared. Although the lenses of kfrs4/kfrs4 homozygotes showed severely disorganised fibres and vacuolation, the lenses of kfrs4/+ heterozygotes appeared normal and similar to those of wild-type rats. We used positional cloning to identify the kfrs4 mutation. The mutation was mapped to an approximately 9.7-Mb region on chromosome 7, which contains the Mip gene. This gene is responsible for a dominant form of cataract in humans and mice. Sequence analysis of the mutant-derived Mip gene identified a 5-bp insertion. This insertion is predicted to inactivate the MIP protein, as it produces a frameshift that results in the synthesis of 6 novel amino acid residues and a truncated protein that lacks 136 amino acids in the C-terminal region, and no MIP immunoreactivity was observed in the lens fibre cells of kfrs4/kfrs4 homozygous rats using an antibody that recognises the C- and N-terminus of MIP. In addition, the kfrs4/+ heterozygotes showed reduced expression of Mip mRNA and MIP protein and the kfrs4/kfrs4 homozygotes showed no expression in the lens. These results indicate that the kfrs4 mutation conveys a loss-of-function, which leads to functional inactivation though the degradation of Mip mRNA by an mRNA decay mechanism. Therefore, the kfrs4 rat represents the first characterised rat model with a recessive mutation in the Mip gene.     

Mechanisms of modulation of neuronal nicotinic receptors by substance P and OAG

Substance P is known to modulate neuronal nicotinicacetylcholine receptors (nAChRs) in the sympathetic nervous system.There are two conflicting proposals for the mechanism of this effect, an indirect action mediated by protein kinase C (PKC) and a direct interaction with receptor subunits. We studied the mechanisms of thiseffect in PC-12 cells. Substance P enhanced the decay of thenicotine-induced whole cell current. This effect was fast in its onsetand was not antagonized by guanosine5'-O-(2-thiodiphosphate), a G protein blocker, orstaurosporine, a nonselective PKC blocker. Staurosporine failed toreverse the inhibition by 1-oleoyl-2-acetyl-sn-glycerol (OAG), a synthetic diacylglycerol analog known to activate PKC. Theinhibitory effects of the peptide and OAG were preserved in excisedpatches, but substance P applied to the extra patch membrane wasineffective in the cell-attached patch configuration. We conclude thatsubstance P modulates neuronal nAChRs most likely by direct interactions with the receptors but independently from activation ofPKC or G proteins and that PKC does not participate in modulation by OAG.

     

Purification and characterization of thymidylate synthetase from rat regenerating liver

Thymidylate synthetase (EC 2.1.1.45) from rat regenerating liver has been purified over 5000-fold to apparent homogeneity by a procedure involving two affinity methods. Molecular weight of the native enzyme was found to be about 68,000, as determined by gel filtration. Electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate yielded a single band of molecular weight of 35,000, suggesting that thymidylate synthetase is a dimer of very similar or identical subunits. The Michaelis constants for deoxyuridylate (dUMP) and (+/-)L-5,10-methylenetetrahydrofolate are 6.8 microM and 65 microM, respectively. Reaction kinetics and product inhibition studies reveal the enzymatic mechanism to be ordered sequential. 5-Fluoro-dUMP, halogenated analog of the nucleotide substrate is a competitive inhibitor of the enzyme, with an apparent Ki value of 5 nM. Amethopterin, analog of the cofactor is also a competitive inhibitor with an apparent Ki value of 23 microM.     

Simulating Evolution by Gene Duplication

By considering the recent finding that unequal crossing over and other molecular interactions are contributing to the evolution of multigene families, a model of the origin of repetitive genes was studied by Monte Carlo simulations. Starting from a single gene copy, how genetic systems evolve was examined under unequal crossing over, random drift and natural selection. Both beneficial and deteriorating mutations were incorporated, and the latter were assumed to occur ten times more frequently than the former. Positive natural selection favors those chromosomes with more beneficial mutations in redundant copies than others in the population, but accumulation of deteriorating mutations (pseudogenes) have no effect on fitness so long as there remains a functional gene. The results imply the following: Positive natural selection is needed in order to acquire gene families with new functions. Without it, too many pseudogenes accumulate before attaining a functional gene family. There is a large fluctuation in the outcome even if parameters are the same. When unequal crossing over occurs more frequently, the system evolves more rapidly. It was also shown, under realistic values of parameters, that the genetic load for acquiring a new gene is not as large as J.B.S. Haldane suggested, but not so small as in a model in which a system for selection started from already redundant genes.     

Effect of colchicine and vincristine on DNA synthesis in regenerating rat liver

The increases in the activities of hepatic thymidylate synthetase and thymidine kinase were significantly suppressed at 24 h after 70% partial hepatectomy in rats which had been administered a microtubule disrupter, colchicine or vincristine. The decrease of these enzymic activities was accompanied by a reduction of DNA content in 24 h regenerating liver. The immunoblotting assay showed that the depression of the thymidylate synthetase activity by the injection of colchicine or vincristine was due to the decrease of the enzyme protein. These results indicate that colchicine and vincristine inhibit the DNA synthesis during liver regeneration by inhibiting the induction of the key enzyme in DNA synthesis.