Saburo Omata

Barrier-to-autointegration factor plays crucial roles in cell cycle progression and nuclear organization in Drosophila

Barrier-to-autointegration factor (BAF) is potentially a DNA-bridging protein, which directly associates with inner nuclear membrane proteins carrying LEM domains. These features point to a key role in regulation of nuclear function and organization, dependent on interactions between the nuclear envelope and chromatin. To understand the functions of BAF in vivo, Drosophila baf null mutants generated by P-element-mediated imprecise excision were analyzed. Homozygous null mutants showed a typical mitotic mutant phenotype: lethality at the larval-pupal transition with small brains and missing imaginal discs. Mitotic figures were decreased but a defined anaphase defect as reported for C. elegans RNAi experiments was not observed in these small brains, suggesting a different phase or phases of cell cycle arrest. Specific abnormalities in interphase nuclear structure were frequently found upon electron microscopic examination of baf null mutants, with partial clumping of chromatin and convolution of nuclear shape. At the light microscopic level, grossly aberrant nuclear lamina structure and B-type lamin distribution correlated well with the loss of detectable amounts of BAF protein from nuclei. Together, these data represent evidence of BAFs anticipated function in mediating interactions between the nuclear envelope and interphase chromosomes. We thus conclude that BAF plays essential roles in nuclear organization and that these BAF functions are required in both M phase and interphase of the cell cycle.

Identification of the Site of Interaction of the 14-3-3 Protein with Phosphorylated Tryptophan Hydroxylase

The 14-3-3 protein family plays a role in a wide variety of cell signaling processes including monoamine synthesis, exocytosis, and cell cycle regulation, but the structural requirements for the activity of this protein family are not known. We have previously shown that the 14-3-3 protein binds with and activates phosphorylated tryptophan hydroxylase (TPH, the rate-limiting enzyme in the biosynthesis of neurotransmitter serotonin) and proposed that this activity might be mediated through the COOH-terminal acidic region of the 14-3-3 molecules. In this report we demonstrate, using a series of truncation mutants of the 14-3-3 isoform expressed in Escherichia coli, that the COOH-terminal region, especially restricted in amino acids 171-213, binds indeed with the phosphorylated TPH. This restricted region, which we termed 14-3-3 box I, is one of the structural regions whose sequence is highly conserved beyond species, allowing that the plant 14-3-3 isoform (GF14) could also activate rat brain TPH. The 14-3-3 box I is the first functional region whose activity has directly been defined in the 14-3-3 sequence and may represent a common structural element whereby 14-3-3 interacts with other target proteins such as Raf-1 kinase. The result is consistent with the recently published crystal structure of this protein family, which suggests the importance of the negatively charged groove-like structure in the ligand binding.

In vivo effect of methylmercury on protein synthesis in brain and liver of the rat

Abstract Rats were given daily sc injections of methylmercury chloride, 10 mg/kg for 7 consecutive days. The manifestation of the neurological syndrome in the rat and the accumulation of mercury in rat tissues resembled the observations of previous investigators. The incorporation in vitro of [ 14 C]leucine into brain protein began to decrease during the latent period of intoxication and declined to 56% of the control values at the symptomatic period. The incorporation of [ 14 C]leucine into liver protein was also inhibited to a similar extent at the symptomatic period following a remarkable stimulation at the early stage after the onset of administration of methylmercury. The impairment of protein synthesis in the brain and liver at the symptomatic period was confirmed by the incorporation in vivo of a 14 C-labeled amino acid mixture into proteins of these tissues. The decrease in the [ 14 C]leucine incorporation in the liver of poisoned rats was largely affected by nutritional deficiency due to decreased food intake, but that in the brain resulted from the direct effect of methylmercury on this tissue.

Time-dependent accumulation of inorganic mercury in subcellular fractions of kidney, liver, and brain of rats exposed to methylmercury

Accumulation of inorganic mercury in subcellular fractions of the kidney, liver, and brain of rats was studied during 48 days after a single injection of 25 mg/kg of methylmercury chloride. The highest ratio of inorganic to total mercury was seen in the cytosol of kidney, 80% of the total being as inorganic mercury at day 48. The ratio in the mitochondria and microsomes of kidney attained a maximum level (about 50% of the total as inorganic) at day 26–37. In the liver, the ratio was strikingly low in the cytosol and microsomes as compared to the light and heavy mitochondria where about 40% of the total was present as inorganic maximally at day 26. The ratio in the brain, determined up to day 15, was very low as compared with the kidney and liver, showing less than 3% of the total in the mitochondria, microsomes, and cytosol, and 5.4% in the myelin fraction. The high accumulation of inorganic mercury in the cytosol of kidney was closely related to metallothionein-like component, while those in the mitochondria and microsomes of kidney and in the mitochondria of liver were exclusively bound to high molecular weight proteins even after deoxycholate treatment.

In vivo effect of methylmercury on protein synthesis in peripheral nervous tissues of the rat.

The in vivo rates of protein synthesis in the peripheral nervous tissues of methylmercury-treated rats (10 mg/kg/day, for 7 days) have been estimated with improved methods by the injection of a large amount of [l-14C]valine of low specific activity. Protein synthesis activity in the dorsal root ganglia was inhibited to the extent of 60% of the control as early as day 5 and this continued to the symptomatic period (day 15) on which crossing of hind limbs, a typical sign of organomercurial poisoning, was observed in the animals. The sciatic nerves and dorsal roots increased protein synthesis by 56% at the symptomatic period. These increases in protein synthesis may be due to the stimulation of reactivity of Schwanns cells. On the contrary, the protein synthesis in the ventral roots showed a gradual decrease as the intoxication proceeded and decreased to 73% of the control at the symptomatic period, being similar to the case of the brain. The double-labeling studies with sodium dodecyl sulfate/polyacrylamide gel electrophoresis exhibited that methylmercury inhibited the synthesis of the dorsal root ganglion proteins non-uniformly in various apparent molecular sizes, especially on day 10.

In vitro nuclear assembly with affinity-purified nuclear envelope precursor vesicle fractions, PV1 and PV2.

Nuclear envelope precursor vesicles were affinity purified from a Xenopus egg extract by a chromatin binding method. Vesicles bound to chromatin at 4 degrees C were dissociated with a high salt buffer and further fractionated into nuclear envelope precursor vesicle fractions 1 (PV1) and 2 (PV2) by differential centrifugation. PV1 contained larger vesicles. When chromatin was incubated in a Xenopus egg cytosol fraction supplemented with PV1, vesicles bound to chromatin, fused with each other, formed a bilayered nuclear envelope, and assembled into spherical small nuclei. However, the thus assembled nuclei did not grow to the normal size. Nuclear pore complexes were not found on the thus assembled nuclei. On the other hand, PV2 contained smaller vesicles. PV2 vesicles bound to chromatin, fused little with each other in the Xenopus egg cytosol fraction, and no nuclei were assembled. When PV1 supplemented with PV2 was used for the nuclear assembly reaction, the assembled nuclei grew to the normal size. Nuclear pore complexes existed in the thus assembled nuclear envelopes. These results suggested that 1) two vesicle populations, PV1 and PV2, are necessary for the assembly of normal sized nuclei, 2) PV1 contains a chromatin targeting molecule(s) and membrane fusion machinery, 3) PV2 contains a chromatin targeting molecule(s) and a molecule(s) necessary for nuclear pore complex assembly, and 4) PV1 has the ability to assemble a nuclear membrane, and PV2 is necessary for the assembly of nuclear pore complexes and for nuclei to grow to the normal size. An in vitro nuclear assembly system constituted with affinity-purified vesicle fractions, PV1 and PV2, was established.

Methylmercury-induced changes in the activities of neurotransmitter enzymes in nervous tissues of the rat

The activities of neurotransmitter-metabolizing enzymes were determined in the nervous tissues of rats treated with methylmercury chloride (10 mg/kg/day, for 7 days). The activity of choline acetyltransferase was lowered consistently in the cytosol and synaptosomal fractions of the brain in methylmercury-treated rats, while changes in acetylcholinesterase activity in the brain subcellular fractions were small. In peripheral nerves, decreases in activities of choline acetyltransferase in the sciatic nerve and of acetylcholinesterase in the dorsal root were most profound. Decreases in these enzyme activities started at an early phase and increased markedly with the progress of intoxication. The activity of acetylcholinesterase in the dorsal root ganglion and in the sciatic nerve was also inhibited significantly at the latent period and more profoundly at the symptomatic period at which time crossing of hind limbs, a typical sign of organomercurial poisoning, was observed in the animals. Activities of tyrosine hydroxylase and monoamine oxidase were elevated in the brain homogenate and especially in the synaptosomal fraction with respect to the former enzyme after methylmercury treatment. Effects of methylmercury in vitro on the activities of these enzymes revealed that a much higher amount of methylmercury was required to produce in vitro an inhibitory action equivalent to that observed in vivo. These results suggest that the neurotoxic action of methylmercury could be mediated, at least in part, through the level of neurotransmitter enzymes.

Effect of methylmercury chloride on the in vivo rate of protein synthesis in the brain of the rat: Examination with the injection of a large quantity of [14C]valine

Abstract The in vivo rates of protein synthesis in brains of control and methylmercury-treated rats were estimated by single injections of large quantities of low specific activity [U- 14 C]valine. The specific activities of free valine and protein-bound valine were determined to obtain the average rate of protein synthesis. The average rate in the cerebral cortex of the control rats was 0.75% replacement of protein-bound valine per hour. Methylmercury treatment decreased protein synthesis rates by 32% at the latent phase and by 15% at the symptomatic phase. In other brain regions studied, protein synthesis rates were also inhibited by methylmercury to various extent. The use of [U- 14 C]valine revealed that valine catabolism in the brain of control rats is different from that of methylmercury-treated animals.

Decrease in protein phosphorylation in central and peripheral nervous tissues of methylmercury-treated rat

The protein phosphorylation in extracts of nervous tissues of rats acutely exposed to methylmercury chloride (seven daily injections of 10 mg methylmercury chloride/kg body weight) was examined. In the brain, the phosphorylating activity was dependent on cAMP and Mg2+. The effect of methylmercury on the phosphorylation of brain proteins, including tubulin and MAP-2, was hardly discernible. In peripheral nervous tissues such as the dorsal and ventral roots, sciatic nerves and dorsal root ganglia, the phosphorylating activity was dependent on Ca2+, and the maximal activity was obtained when the tissues were extracted in the presence of 1% Triton X−100. SDS-Polyacrylamide gel electrophoresis revealed that the major phosphorylated proteins in the peripheral tissues were myelin proteins. The effects of methylmercury were not uniform regarding protein species and tissues. The most marked changes were observed in sciatic nerves, in which phosphorylation of the 33 kDa, 28 kDa, 19 kDa, 18 kDa and 15 kDa proteins was significantly decreased in the symptomatic phase of intoxication.

Isolation and some properties of a 34‐kDa‐membrane protein that may be responsible for ribosome binding in rat liver rough microsomes

We have isolated, by hydroxyapatite chromatography with a non ionic detergent and a high salt concentration, a non‐glycosylated, membrane protein with a relative molecular weight of 34 kDa that had previously been found to be a major constituent of the membrane protein fraction showing ribosoine‐binding activity derived from rat liver rough microsomes (RM). The isolated 34 kDa protein (p34), when incorporated into a liposome model membrane, exhibited significant binding activity toward ribosomes, its binding properties being similar to those observed with intact R.M. immunochemical analyses using antibodies directed against p34 suggested that it is a membrane‐embedded RM surface protein, which is specifically localized in ribosome‐attached organelles and widely distributed among mammalian tissues. These results would constitute evidence that p34 is a likely candidate for an RM ribosome‐binding, protein.