Synthesis of sex-specific forms of cytochrome P-450 in rat liver is transiently suppressed by hepatic monooxygenase inducers

The effects of phenobarbital (PB), 3-methylcholanthrene (MC), and alpha-naphthoflavone (alpha-NF) on the synthesis of drug-inducible forms of cytochrome P-450, P-450(PB-1), and P-450(MC-1), and sex-specific forms of cytochrome P-450, P-450(M-1), and P-450(F-1), in male and female rats were studied. Whereas P-450(PB-1) and P-450(MC-1) in liver microsomes were markedly induced in both sexes by treatment with PB and MC, respectively, the contents of P-450(M-1) and P-450(F-1) were significantly decreased by the treatments. alpha-NF, which is not a P-450 inducer, did not change the contents of sex-specific forms of cytochrome P-450. The translatable mRNAs of the P-450s were also determined by using an in vitro translation system. The mRNAs coding for P-450(PB-1) and P-450(MC-1) were increased by drug administrations. On the other hand, the mRNAs coding for P-450(M-1) and P-450(F-1) were transiently decreased by the drugs, and then returned to the normal levels. The time courses of the induction of the drug-inducible P-450s and the repression of the sex-specific P-450s showed no close correlation. alpha-NF had no effect on the synthesis of P-450(M-1) and P-450(F-1). We also found that the synthesis of P-450(M-1) in the livers of untreated rats showed no diurnal variations.     

Different requirement for cytochrome b5 in NADPH-supported O-deethylation of p-nitrophenetole catalyzed by two types of microsomal cytochrome P-450

The role of cytochrome b₅ in the NADPH-supported O-deethylation of p-nitrophenetole catalyzed by cytochrome P-450 was studied with reconstituted systems using two types of cytochrome P-450 (P-450_PB and P-450_MC) purified from rat liver microsomes. The O-deethylation by P-450_PB absolutely required the presence of cytochrome b₅, whereas the same reaction catalyzed by P-450_MC did not require cytochrome b₅. These effects of cytochrome b₅ on the activities of reconstituted systems were confirmed by the use of antibodies to cytochrome b₅. On the other hand, the oxidations of ethylmorphine and aniline by these two types of cytochrome P-450 did not show significant dependence on cytochrome b₅. These observations suggest that the requirement for cytochrome b₅ in NADPH-supported drug oxidations depends not only on the species of cytochrome P-450 catalyzing the reactions, but also on the substrates oxidized.     

Golgin-84 is a rab1 binding partner involved in Golgi structure

Members of the golgin family of coiled-coil proteins have been implicated in the tethering of vesicles to Golgi membranes and cisternal membranes to each other. Many also bind to rab GTPases. Golgin-84 is a membrane-anchored golgin that we now show binds preferentially to the GTP form of the rab1 GTPase. It is also present throughout the Golgi stack by immuno-EM. Antibodies to golgin-84 inhibit stacking of cisternal membranes in a cell-free assay for Golgi reassembly, whereas the cytoplasmic domain of golgin-84 stimulates stacking and increases the length of re-assembled stacks. Transient expression of golgin-84 in NRK cells helps prevent the disassembly of the Golgi apparatus normally triggered by treatment with brefeldin A. Together these data suggest that golgin-84 is involved in generating and maintaining the architecture of the Golgi apparatus.     

In vitro effects of L-ascorbic acid (vitamin C) on aryl hydrocarbon hydroxylase activity in hepatic microsomes of mice.

When aromatic hydrocarbon (Ah)-responsive and -non-responsive strains of mice were pretreated with 3-methylcholanthrene (MC) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), vitamin C reduced the microsomal aryl hydrocarbon hydroxylase (AHH) activity. The AHH inhibitors 7,8-benzoflavone (7,8-BF) and 3-methylsulfonyl-3',4,4',5-tetrachlorobiphenyl (3-MSF-3',4,4',5-tetraCB) showed various inhibitory effects depending upon the types of microsomes, whereas vitamin C exhibited inhibition irrespective of the types of microsomes. 7,8-BF and 3-MSF-3',4,4',5-tetraCB as well as vitamin C suppressed the reverse mutation of the Salmonella typhimurium tester strains TA98 and TA100 induced by benzo[a]pyrene.     

Construction and expression of anti-Tn-antigen-specific single-chain antibody genes from hybridoma producing MLS128 monoclonal antibody

Anti-Tn-antigen monoclonal antibody MLS128 has affinity for three consecutive Tn-antigens (Tn3) more than Tn2. The major aim of this study was to isolate genes encoding MLS128 variable domains to produce a large quantity of recombinant MLS128 antibodies, in turn, allowing the conduct of studies on precise interactions between Tn3- or Tn2-epitopes and MLS128. This study describes cloning of the variable region genes of MLS128, construction of the variable region genes in single-chain variable fragments (scFv) and two scFvs conjugated with human IgG(1) hinge and Fc regions (scFv-Fc) types, and their respective expression in bacterial and mammalian cell. MLS128 scFv protein with the expected specificity and affinity was successfully prepared from inclusion bodies accumulating in Escherichia coli. Construction, expression and purification of two types of MLS128-scFv-Fc proteins with differing linker lengths in Chinese hamster ovary cells demonstrated that the purified scFv-Fc proteins had binding activity specific to the glycoprotein-expressing Tn-antigen clusters. These results revealed that VL and VH genes cloned from the hybridoma represent those of MLS128 and that recombinant antibodies produced from these genes should provide sufficient amounts of binding domains for use in 3D structural studies such as NMR and X-ray analysis.     

Cytosolic and mitochondrial surface factor-independent import of a synthetic peptide into mitochondria.

We chemically synthesized a peptide, 11 beta-45, which was composed of 45 amino acid residues including the whole extension peptide and some of the mature portion of bovine cytochrome P-450(11 beta) precursor. 11 beta-45 was imported into mitochondria in vitro depending on the mitochondrial membrane potential, but its import did not require extramitochondrial ATP. Although cytosolic protein factors in the high speed supernatant of reticulocyte lysate are known to stimulate the import of various precursor proteins into mitochondria, the import of 11 beta-45 was not stimulated by cytosolic factors in reticulocyte lysate. The import of the peptide did not require mitochondrial surface protein components because its import was not affected by trypsin treatment of mitochondria. On the other hand, trypsin treatment of mitoplasts resulted in a great reduction in the import of the peptide, indicating that 11 beta-45 interacts during the import process with some protein components located inside mitochondria. These observations indicated that the peptide 11 beta-45 was imported via the potential-dependent pathway as in the case of precursor proteins, but skipped the interactions with cytosolic factors and mitochondrial surface components normally required for the import of precursor proteins.     

Age-related changes of elements and relationships among elements in the common bile and pancreatic ducts

To elucidate compositional changes of the common bile and main pancreatic ducts with aging, the authors investigated age-related changes of element contents in the common bile and pancreatic ducts by inductively coupled plasma-atomic emission spectrometry. After ordinary dissection by medical students was finished, the common bile ducts and main pancreatic ducts (pancreatic ducts) were resected and the element contents were determined. The Mg content increased significantly only in the pancreatic duct with aging, but the other element contents did not change significantly in both the common bile and pancreatic ducts with aging. Regarding the relationship among the elements, significant direct correlations were found among the contents of Ca, P, S, and Mg in the common bile ducts, with some exceptions between P and either S or Mg contents. In the pancreatic ducts, significant direct correlations were found between S and Mg contents and between P and Na contents. The relationships in the elements between the common bile and pancreatic ducts were examined. It was found that there were significant direct correlations in the Ca, Mg, and Fe contents between the common bile and pancreatic ducts; that is, as Ca, Mg, and Fe increased in the common bile duct, they increased simultaneously in the pancreatic duct.     

Isolation of a bacterium that causes anaaki disease of the red algae Porphyra yezoensis

Anaaki disease causes severe damage to the red algae Porphyra yezoensis from which the Japanese traditional food 'nori'is produced. The causative agent of anaaki disease was isolated by several repeats of single-colony isolation and infection experiments, and was identified as Flavobacterium sp. LAD-1. The bacterium showed hydrolytic activity toward porphyran but not toward other polysaccharides composing the thallus of Porphyra , such as β-1,3-xylan or β-1,4-mannan. The bacterium also showed β-D-galactosidase activity.     

Phosphorylation of p66shc mediates 6-hydroxydopamine cytotoxicity

6-Hydroxydopamine (6-OHDA) is a neurotoxin that has been widely used to generate Parkinson's disease (PD) models. Increased oxidative stress is suggested to play an important role in 6-OHDA-induced cell death. Given the lessened susceptibility to oxidative stress exhibited by mice lacking p66shc, this study investigated the role of p66shc in the cytotoxicity of 6-OHDA. 6-OHDA induced cell death and p66shc phosphorylation at Ser36 in SH-SY5Y cells. Pre-treatment with the protein kinase C β (PKCβ) inhibitor hispidin suppressed 6-OHDA-induced p66shc phosphorylation. Elimination of H(2)O(2) by catalase reduced cell death and p66shc phosphorylation induced by 6-OHDA. Cells deficient in p66shc were more resistant to 6-OHDA-induced cell death than wild-type cells. Furthermore, reconstitution of wild-type p66shc, but not the S36A mutant, in p66shc-deficient cells increased susceptibility to 6-OHDA. These results indicate that H(2)O(2) derived from 6-OHDA is an important mediator of cell death and p66shc phosphorylation induced by 6-OHDA and that p66shc phosphorylation at Ser36 is indispensable for the cytotoxicity of 6-OHDA.