Toshihiro Sassa

Vitamin B6 deficiency causes activation of RNA polymerase and general enhancement of gene expression in rat liver

The effect of vitamin B6 deficiency on the activity of RNA polymerase and expression of several mRNAs in rat liver was investigated. The activities of RNA polymerase I and II in the liver of vitamin B6‐deficient rats were found to be higher than the control rats by 30%. The expression of several mRNAs, including mRNAs for β‐actin and glyceraldehyde‐3‐phosphate dehydrogenase, and the content of poly(A)+ RNA were also increased in vitamin deficiency. These observations suggest that vitamin B6 influences gene expression in the liver, at least in part, by modulating the activity of RNA polymerase.

The synaptic protein UNC-18 is phosphorylated by protein kinase C.

The C. elegans unc-18 encoded protein UNC-18 is implicated in the interactions between synaptic vesicles and presynaptic plasma membrane. To further characterize the neural protein, we investigated the phosphorylation in vitro of the protein expressed in Spodoptera frugiperda Sf21 cells. The UNC-18 protein is selectively phosphorylated by protein kinase C (PKC) but not by casein kinase II and cyclic AMP-dependent protein kinase. The presumed phosphorylation sites determined by manual Edman degradation were serine-2, serine-322, threonine-462 and serine-515, of which the last is highly conserved as a consensus phosphorylation site for PKC in Drosophila and the mammalian homologue. Phosphorylated UNC-18 extracted from C. elegans was also detected, indicating that it has a physiological role in intact nerve terminals. Therefore, the phosphorylation by PKC may play a physiological role in the regulation.

Endocytosis of Serum Albumin in Regenerating Rat Liver

Abstract Lysosomes, isolated from rat liver after 70% partial hepatectomy (PHX), were found, by Western blotting, to contain a considerable amount of serum albumin. The level of intralysosomal serum albumin after PHX showed biphasic patterns: it increased immediately after PHX, peaked at 30 min, rapidly declined within a few hours, rose again with a peak at 15 hr, and gradually declined thereafter. At 15 hr after PHX, the content of lysosomal proteins in the liver increased to twice the level of unoperated control, and the electron-microscopic observation of the isolated lysosomes revealed numerous large membrane-delimited structures with ground substances of variable electron opacities. The increase in the intralysosomal serum albumin at 30 min and 15 hr was accompanied by changes in the buoyant densities of endosomes in Percoll density gradients. At both time points, the density profiles of endosomes isolated from hepatectomized rats shifted to the denser direction, suggesting that PHX activates fusion and/or maturation of endosomes. Formaldehyde-treated bovine serum albumin is known to be taken up by the liver by receptor-mediated endocytosis. The uptake of the modified heterologous albumin was shown to be activated as early as 30 min after PHX. Both the uptake of serum albumin into lysosomes and the shift of buoyant density profile of endosomes after PHX were inhibited by the administration of adrenergic receptor antagonists, particularly by the α1-antagonist prazosin. Further, the concentration of catecholamines in rat serum, particularly that of norepinephrine, was found to increase immediately after PHX, relative to that in serum from sham-operated rats. These results suggest that the elevation of serum norepinephrine levels after PHX activates endocytosis and facilitates delivery of endocytosed serum albumin to lysosomes, where albumin is digested to yield amino acids for possible use in protein synthesis during liver regeneration.

Developmental changes in the expression of HMG 2a protein

The levels of HMG 2a chromosomal protein and its mRNA change during the post‐hatched development of chicks were investigated. The contents of both HMG 2a and 2b proteins of liver, heart, brain, muscle and gizzard were abundant in the newly hatched chicks but their contents decreased significantly in those tissues of the 70‐day‐old chicks. The HMG 2a mRNA levels of liver, heart and brain in 70‐day‐old chick decreased to about 40% of those mRNA in the newly hatched chicks while the HMG 2a mRNA levels of muscle and gizzard in the 70‐day‐old chicks increased 5‐ and 3‐fold, respectively. These results suggest that the decrease in the HMG 2a protein contents of the muscle and gizzard in the 70‐day‐old chicks may be largely due to the stimulation of HMG 2a protein degradation or the reduction of HMG 2a mRNA translation.

INTERACTION OF RAT LIVER LYSOSOMES WITH BASIC POLYPEPTIDES

In order to gain knowledge on the interaction of lysosomes with proteins, we have assessed the equilibrium densities of the lysosomal membrane and matrix markers after in vitro incubation of rat liver lysosomes with various polypeptides. The addition of basic polypeptides, polylysine or protamine, to the suspension of lysosomes brought about a profound alteration of lysosomal membrane, causing extensive leakage of lysosomal matrix enzymes. Electron microscopic observation revealed a remarkable aggregation of lysosomes by the basic polypeptides. Polyglutamic acid, an acidic polypeptide, did not produce such effect. ATP was found to stabilize lysosomes during incubation, particularly with basic polypeptides.

Production of functional chick liver HMG 2a protein in Escherichia coli

An efficient Escherichia coli system for the production of a variant form of high‐mobility group‐2a protein (HMG 2a), having the additional 5 amino acid residues (Ala‐Pro‐Thr‐Leu‐Glu) at the NH2‐terminal, has been constructed. cDNA encoding HMG 2a was ligated with the Omp A signal peptide sequence and was inserted into an inducible bacterial expression vector pSH‐L. After the plasmid introduced into E. coli was expressed by temperature shift, the recombinant product was purified by trichloacetic acid precipitation followed by Bio‐Rex 70 column chromatography. The purified product showed the expected NH2‐terminal sequence and the superhelical activity of circular DNA similar to the authentic HMG 2a isolated from chick liver.

Phenylmethylsulfonyl fluoride stimulates proteolysis of nuclear proteins from chick liver

The degradation of H1 histone and high mobility group (HMG) nonhistone proteins was stimulated when the homogenate from chick liver was incubated in the presence of phenylmethylsulfonyl fluoride (PMSF). Two proteinase inhibitors, elastatinal and chymostatin, significantly inhibited the PMSF-stimulated degradation of H1 histone and HMG proteins. On the contrary, other proteinase inhibitors like leupeptin, pepstatin, trypsin inhibitor, antipain, o-phenanthroline and EDTA had no effect on the degradation of the nuclear proteins. These results warn the researcher to be cautious while using PMSF for preparation of nuclear proteins such as H1 histone and HMG proteins.