Mdm20 Modulates Actin Remodeling through the mTORC2 Pathway via Its Effect on Rictor Expression

NatB is an N-terminal acetyltransferase consisting of a catalytic Nat5 subunit and an auxiliary Mdm20 subunit. In yeast, NatB acetylates N-terminal methionines of proteins during de novo protein synthesis and also regulates actin remodeling through N-terminal acetylation of tropomyosin (Trpm), which stabilizes the actin cytoskeleton by interacting with actin. However, in mammalian cells, the biological functions of the Mdm20 and Nat5 subunits are not well understood. In the present study, we show for the first time that Mdm20-knockdown (KD), but not Nat5-KD, in HEK293 and HeLa cells suppresses not only cell growth, but also cellular motility. Although stress fibers were formed in Mdm20-KD cells, and not in control or Nat5-KD cells, the localization of Trpm did not coincide with the formation of stress fibers in Mdm20-KD cells. Notably, knockdown of Mdm20 reduced the expression of Rictor, an mTORC2 complex component, through post-translational regulation. Additionally, PKCα^S657 phosphorylation, which regulates the organization of the actin cytoskeleton, was also reduced in Mdm20-KD cells. Our data also suggest that FoxO1 phosphorylation is regulated by the Mdm20-mTORC2-Akt pathway in response to serum starvation and insulin stimulation. Taken together, the present findings suggest that Mdm20 acts as a novel regulator of Rictor, thereby controlling mTORC2 activity, and leading to the activation of PKCα^S657 and FoxO1.     

Genetic analysis of a mutation affecting ribosomal protein S1 in Escherichia coli

Molecular Genetics and Genomics - Ribosomal protein S1 from a newly isolated Escherichia coli mutant has a molecular weight of about 54,000 which is smaller than the wild type S1 (M.W. 65,000). The...     

Purification and Properties of Prostaglandin H-E Isomerase from the Cytosol of Human Brain: Identification as Anionic Forms of Glutathione S-Transferase

Abstract: Prostaglandin H-E isomerase (EC 5.3.99.3) was purified from human brain cytosol. Purification was by ammonium sulfate fractionation, diethylaminoethyl-Sephar-ose chromatography, gel filtration on a BioGel P-100 column, GSH-agarose chromatography, and MonoQ chromatography. The activity was eluted in two peaks from the MonoQ column, which were designated peaks 1 and 2. The molecular weights of peaks 1 and 2, determined by gel filtration, were 42,000 and 44,000, respectively. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, peak 1 showed two bands at the molecular weights of 24,500 and 25,000, and peak 2 showed a single band at the molecular weight of 25,000, results suggesting that both were dimeric proteins. The pI values of both enzymes were ∼5.4. The enzymes catalyzed selective conversion of prostaglandin H₂ to prostaglandin E₂. The K _m values for prostaglandin H₂ of peaks 1 and 2 were 147 and 308 μ M , respectively, and the V _max values were 380 and 720 nmol/min/mg of protein, respectively. GSH was required for the catalysis of both enzymes, and no other sulfhydryl compounds could support the reaction. A part of glutathione S -transferase (EC 2.5.1.18) was copurified with peaks 1 and 2 of prostaglandin H-E isomerase. Prostaglandin H-E isomerase activity of peak 2 enzyme was competitively inhibited by 1-chloro-2,4-dinitrobenzene, a substrate of glutathione S -transferase. These results suggested that prostaglandin H-E isomerases in human brain cytosol were identical with anionic forms of glutathione S -transferase.     

Yeast plasmids resembling 2 micron DNA: regional similarities and diversities at the molecular level.

The nucleotide sequence of two Zygosaccharomyces plasmids, pSB2 (5,415 base pairs), isolated from Zygosaccharomyces bailii, and pSM1 (5,416 base pairs), isolated from Zygosaccharomyces fermentati Naganishi, was determined. The predicted amino acid sequences of open reading frames among six yeast plasmids that resemble 2 microns DNA indicated regional sequence similarities among FLP proteins. Greater similarities were seen among Zygosaccharomyces plasmids (pSB2, pSB3, pSR1, and pSM1) than other combinations. A putative recognition site for the FLP enzyme of a Zygosaccharomyces plasmid also showed some conservation, especially in the 4 nucleotides flanking the central spacer region. From comparative studies of the sequences of putative genes of each plasmid, we propose an apparent phylogenetic relationship among yeast plasmids resembling 2 micron DNA. Among the Zygosaccharomyces plasmids, pSB2 and pSR1 are most closely related, since not only were the FLP enzymes of these two plasmids most closely related, but also the amino acid sequence of the putative P gene of pSR1 showed clear homology with that of open reading frame B of pSB2.     

Immunohistochemical localization of atrial natriuretic polypeptide (ANP) in human atrial and ventricular myocardiocytes

To date, there have been few immunohistochemical investigations of atrial natriuretic polypeptide (ANP) in human cardiac tissue, especially the ventricles. In this study, myocardial tissue was obtained from two sources: the bilateral atria and ventricles at autopsy; and biopsy tissues from the right auricle and left ventricle of a patient with myocardial infarction undergoing surgery. These tissues were examined by the avidin-biotin immunoperoxidase technique using three kinds of primary ANP-antibodies. ANP-immunoreactivity was observed in the perinuclear region of myocytes of all tissues examined. The intensity of the reaction was stronger in atrial tissue, weaker in ventricular tissue. In the later tissue, the positive-staining myocytes were not part of the pulse-conducting system. Although the tissues we studied were not obtained from normal hearts, our data demonstrates that ANP-reactivity can be detected in ventricular myocytes outside the pulse-conducting system.     

A case of diverticular disease of the colon in a Japanese monkey (Macaca fuscata)

Diverticular disease of the colon was detected in a female Japanese monkey by X-ray examination. The monkey was 15 years old and had been kept under captive conditions for nine years. Lack of appetite and activity, and constipation were observed. The monkey was given fiber-rich vegetables and wild plants, and its appetite and activity then improved. Based on a consideration of various factors, it is suggested that one possible cause of the diverticulosis in this case was a low dietary fiber intake.     

Expression of adrenocorticotropin-releasing hormone precursor gene in placenta and other nonhypothalamic tissues in man

Adrenocorticotropin-releasing hormone (CRH) is a peptide originally isolated from the hypothalamus. Immunocytochemical and RIA studies have revealed that CRH-like peptide is also localized in human nonhypothalamic tissues and some tumors. To see if CRH is synthesized in these nonhypothalamic tissues and tumors, we examined preproCRH mRNA in these tissues by Northern blot analysis using a cloned human preproCRH gene as a probe. PreproCRH mRNA was detected in human hypothalamus, cerebral cortex, adrenal gland, placenta, pheochromocytoma, and thymic carcinoid. The content of preproCRH mRNA in placenta was apparently greater than that in the whole hypothalamus.     

Immunohistochemical localization of atrial natriuretic polypeptide (ANP) in human atrial and ventricular myocardiocytes

Summary To date, there have been few immunohistochemical investigations of atrial natriuretic polypeptide (ANP) in human cardiac tissue, especially the ventricles. In this study, myocardial tissue was obtained from two sources: the bilateral atria and ventricles at autopsy; and biopsy tissues from the right auricle and left ventricle of a patient with myocardial infarction undergoing surgery. These tissues were examined by the avidin-biotin immunoperoxidase technique using three kinds of primary ANP-antibodies. ANP-immunoreactivity was observed in the perinuclear region of myocytes of all tissues examined. The intensity of the reaction was stronger in atrial tissue, weaker in ventricular tissue. In the later tissue, the positive-staining myocytes were not part of the pulse-conducting system. Although the tissues we studied were not obtained from normal hearts, our data demonstrates that ANP-reactivity can be detected in ventricular myocytes outside the pulse-conducting system.