Studies on the Lipoprotein Lipases of Microorganisms

Mucor javanicus IAM 6108 was cultivated aerobically at large scale in the medium containing corn steep liquor 3.0%, soluble starch 1.0%, soybean yuto 1.0% and inorganic salts, and the lipoprotein lipase produced was recovered by addition of ammonium sulfate (0.7 saturation). From this crude preparation, the enzyme was purified about 13 times, through ammonium sulfate fractionation (0~0.4 saturation), precipitation at pH 4.0, ethanol precipitation (80%) and Sephadex G-200 gel filtration. The purified lipoprotein lipase was sedimented as single peak in ultracentrifugal analysis in the presence of 1.0% sodium dodecylsulfate. The enzymatic properties of the purified enzyme was as follows; optimum pH was 7.0, stable pH range was from 5.0 to 7.0, optimum temperature was 40°C, inactivated rapidly above 50°C. The lipoprotein lipase activity was inhibited by 75% and 88% by 10^?2 m taurocholate and 1.0 m NaCl, respectively. ZnCl₂, CuCl₂, Pb(NO₃)₂, and SnCl₂ at 10^?3 m showed complete inhibition. The ratio of lipoprotein lipase to lipase activity was 10 : 1. Lipoprotein lipase activity was dependent on the concentration of blood plasma which could be substituted by bovine serum albumin or egg albumin to a certain degree. The results suggesting the preferential α-fatty acid hydrolysis was obtained.     

Inositol hexakisphosphate kinases promote autophagy

We and other authors have previously reported that increasing cellular diphosphoinositol pentakisphosphate (InsP(7)) levels increases cell sensitivity to cell death. In the present study, we elucidated the relationship between inositol hexakisphosphate kinases (InsP(6)Ks), which form InsP(7), and autophagy using InsP(6)Ks overexpression and disruption systems. A large number of autophagosomes were induced in cells transfected with InsP(6)Ks, as revealed by the conversion of LC3-I to LC3-II, which was examined using immunoblotting, immunocytochemistry, and immuno-electron microscopy for LC3; consequently, the rate of cell death was higher among these cells than among cells transfected with a control vector, as shown using propidium iodide staining. However, the reduction of InsP(6)Ks levels using RNAi suppressed the formation of autophagosomes. Moreover, the number of autophagosomes and the rate of cell death were significantly higher among cells transfected with InsP(6)Ks subjected to staurosporine-induced stress than among cells transfected with InsP(6)Ks subjected to normal conditions. The cell death induced by InsP(6)Ks was not completely suppressed by z-VAD-fmk, a pan-caspase inhibitor. The phosphorylation of mammalian target of rapamycin (mTOR) was also depressed in cells overexpressing InsP(6)Ks, suggesting that the mTOR pathway regulates autophagosomes generated by InsP(6)Ks. These findings imply that InsP(6)Ks promote autophagy and induce caspase-independent cell death. This phenomenon opens a new pathway of autophagy via InsP(6)Ks.     

The Structural Differences between a Glycoprotein Specific F-Box Protein Fbs1 and Its Homologous Protein FBG3

The Skp1-Cul1-F-box protein (SCF) complex catalyzes protein ubiquitination in diverse cellular processes and is one of the best-characterized ubiquitin ligases. F-box proteins determine the substrate specificities of SCF ubiquitin ligases. Among these, Fbs1/FBG1/FBXO2, Fbs2/FBG2/FBXO6, and Fbs3/FBG5/FBXO27 recognize the N-glycans of glycoproteins, whereas FBG3/FBXO44 has no sugar-binding activity, despite the high sequence homology and conservation of the residues necessary for oligosaccharide binding between Fbs1–3 and FBG3. Here we determined the crystal structure of the Skp1–FBG3 complex at a resolution of 2.6 Å. The substrate-binding domain of FBG3 is composed of a 10-stranded antiparallel β-sandwich with three helices. Although the overall structure of FBG3 is similar to that of Fbs1, the residues that form the Fbs1 carbohydrate-binding pocket failed to be superposed with the corresponding residues of FBG3. Structure-based mutational analysis shows that distinct hydrogen bond networks of four FBG3 loops, i.e., β2-β3, β5-β6, β7-β8, and β9-β10, prevent the formation of the carbohydrate-binding pocket shown in Fbs1.     

Sequence analysis of glutamate dehydrogenase (GDH) from the hyperthermophilic archaeon Pyrococcus sp. KOD1 and comparison of the enzymatic characteristics of native and recombinant GDHs

The gdhA gene encoding glutamate dehydrogenase (GDH) from the hyperthermophilic archaeon Pyrococcus sp. KOD1 was cloned and sequenced. Phylogenetic analysis was performed on an alignment of 25?GDH sequences including KOD1-GDH, and two protein families were distinguished, as previously reported. KOD1-GDH was classified as new member of the hexameric GDH Family II. The gdhA gene was expressed in Escherichia coli, and recombinant KOD1-GDH was purified. Its enzymatic characteristics were compared with those of the native KOD1-GDH. Both enzymes had a molecular mass of 47 300?Da and were shown to be functional in a hexameric form (284?kDa). The N-terminal amino acid sequences of native KOD1-GDH and the recombinant GDH were VEIDPFEMAV and MVEIDPFEMA, respectively, indicating that native KOD1-GDH does not retain the initial methionine at the N-terminus. The recombinant GDH displayed enzyme characteristics similar to those of the native GDH, except for a lower level of thermostability, with a half-life of 2?h at 100°?C, compared to 4?h for the native enzyme purified from KOD1. Kinetic studies suggested that the reaction is biased towards glutamate production. KOD1-GDH utilized both coenzymes NADH and NADPH, as do most eukaryal GDHs.     

Cell growth and differentiation in vitro in mouse macrophages transformed by a tsA mutant of simian virus 40. III. Large T antigen level and cell proliferation and survival in an SV40 tsA640-transformed macrophage line

The levels of simian virus 40 (SV40) large T antigen in a tsA-transformed mouse macrophage line at the permissive (33 degrees C) and the nonpermissive (39 degrees C) temperature were examined by immunofluorescence, sodium dodecylsulfate-polyacrylamide gel electrophoresis, complement fixation, and enzyme-linked immunosorbent assay. When the cells were confluent and rested at 33 degrees C, and then were shifted to 39 degrees C, the amount of large T antigen per cell decreased, and most cells survived and remained phagocytic. When the cells were proliferating at 33 degrees C, and then were shifted to 39 degrees C, the cells died with only a small reduction in the amount of large T antigen. Therefore, the physiological state of the cells may determine the survival of cells by affecting the level of large T antigen after exposure to 39 degrees. The confluent cells may be rested with a concomitant decrease of large T antigen. The proliferating cells may not survive in the presence of a relatively high level of functionally defective large T antigen at 39 degrees C.     

Sequential expression of Efhc1/myoclonin1 in choroid plexus and ependymal cell cilia

EFHC1 is a gene mutated in patients with idiopathic epilepsies, and encodes the myoclonin1 protein. We here report the distribution of myoclonin1 in mouse. Immunohistochemical analyses revealed that the myoclonin1 first appeared at the roof of hindbrain at embryonic day 10 (E10), and moved on to choroid plexus at E14. At E18, it moved to ventricle walls and disappeared from choroid plexus. From neonatal to adult stages, myoclonin1 was concentrated in the cilia of ependymal cells at ventricle walls. At adult stages, myoclonin1 expression was also observed at tracheal epithelial cilia in lung and at sperm flagella in testis. Specificities of these immunohistochemical signals were verified by using Efhc1-deficient mice as negative controls. Results of Efhc1 mRNA in situ hybridization were also consistent with the immunohistochemical observations. Our findings raise “choroid plexusopathy” or “ciliopathy” as intriguing candidate cascades for the molecular pathology of epilepsies caused by the EFHC1 mutations.