Molecular cloning of the protocatechuate 4,5-dioxygenase genes of Pseudomonas paucimobilis.

We determined the nucleotide sequence of a 1.9-kilobase fragment of Pseudomonas paucimobilis SYK6 chromosomal DNA that included genes encoding protocatechuate 4,5-dioxygenase, the enzyme responsible for the aromatic ring fission of protocatechuate. Two open reading frames of 417 and 906 base pairs were found that had no homology with previously reported sequences, including those encoding protocatechuate 3,4-dioxygenase. Since both open reading frames were indispensable for the enzyme activity, they should encode the subunits of protocatechuate 4,5-dioxygenase. We named these genes ligA and ligB. Protocatechuate 4,5-dioxygenase was efficiently expressed in Escherichia coli with the aid of the lac promoter, and the polypeptides of the ligA and ligB gene products were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino acid sequencing.     

Developmental Changes in the Bombyxin-and Insulin-like Immunoreactive Neurosecretory System in the Wax Moth, Galleria mellonella

Four medial neurosecretory cells (MNC) and 4 lateral neurosecretory cells (LNC) in each brain hemisphere, and one pair of cells in each thoracic ganglion (TG) of Galleria larva react with antibodies against bombyxin and insulin. Material secreted from the MNC and LNC is released mainly in the corpora allata, and that from the TG through the ventral median nerves. Intrinsic secretory cells of the corpora cardiaca (CC) also contain bombyxin-like, but not insulin-like material. The immunoreactivities all disappear during molts and reappear with resumption of feeding. In the MNC and TG they reappear for less than a day, but in cells of the CC immunoreactivity reappears for the whole feeding period. Before pupation, the LNC become temporarily immunopositive towards the end of feeding period, and the MNC and TG during the wandering period, i.e. at the time of prothoracic gland stimulation. Immunoreactivity disappears during the pupal molt. In pupae it is present in the 4 pairs of MNC and 1–2 pairs of LNC 12–48 hr after ecdysis, and in cells of the CC from 12 hr after ecdysis until the end of the pupal instar. In adult, immunoreactivity is restricted to 2 pairs of the LNC and to CC cells.     

Changes in the levels of prostaglandins and thromboxane and their roles in the accumulation of exudate in rat carrageenin-induced pleurisy — a profile analysis using gas chromatography-mass spectrometry —

Injection of γ-carrageenin into t he pleural cavity of rats caused the accumulation of the pleural exudate. When levels of prostaglandins (PGs) and thromboxane (TX) B₂ were quantified by gas chromatography-mass spectrometry as their methyl ester (ME)-dimethyllisopropylsilyl (DMiPS) ether or ME-methoxine-DMiPS ether derivatives, 6-keto-PGF_1α reached the maximum at 1 hr after carrageenin, then PGE₂ and TXB₂ showed peaks at 3 hr and waned off before 9 hr. he PGF_2α level was kept low, but PGD₂, PGE₁ and PGF_1α were not detected. Aspirin (100 mg/kg, i.p.) significantly decreased the PG and TXB₂ levels and suppressed the rate of plasma exudation until 5 hr, but did not at 7 hr, when it was measured by the amount of exuded pontamine sky blue injected intravenously. OKY-025 (300 mg/kg, i.p.), a selective TXA synthetase inhibitor, and tranylcypromine (20 mg/kg, i.p.), a PGI synthetase inhibitor, could not extensively inhibit the accumulation of the exudate. These results suggest that the cyclooxygenase products of arachidonic acid, particularly PGE₂, definitely play an important role in the exudation during the first 5 hr.     

Regulation of the antioncogenic Chk2 kinase by the oncogenic Wip1 phosphatase

The antioncogenic Chk2 kinase plays a crucial role in DNA damage-induced cell-cycle checkpoint regulation. Here we show that Chk2 associates with the oncogenic protein Wip1 (wild-type p53-inducible phosphatase 1) (PPM1D), a p53-inducible protein phosphatase. Phosphorylation of Chk2 at threonine68 (Thr68), a critical event for Chk2 activation, which is normally induced by DNA damage or overexpression of Chk2, is inhibited by expression of wild-type (WT), but not a phosphatase-deficient mutant (D314A) of Wip1 in cultured cells. Furthermore, an in vitro phosphatase assay revealed that Wip1 (WT), but not Wip1 (D314A), dephosphorylates Thr68 on phosphorylated Chk2 in vitro, resulting in the inhibition of Chk2 kinase activity toward glutathione S-transferase-Cdc25C. Moreover, inhibition of Wip1 expression by RNA interference results in abnormally sustained Thr68 phosphorylation of Chk2 and increased susceptibility of cells in response to DNA damage, indicating that Wip1 acts as a negative regulator of Chk2 in response to DNA damage.     

Interaction of N-ethyl-N'-nitro-n-nitrosoguanidine with nucleic acids and proteins in comparison with N-methyl-N'-nitro-N-nitrosoguanidine

Reactivity of N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) was studied in comparison with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The radioactivity of [guanidino-14C]-ENNG was incorporated only into the protein fraction and that of [ethyl-14C]ENNG was incorporated into DNA, RNA and protein fractions in ascites hepatoma AH7974 cells, as were those of [guanidino-14C]- and [methyl-14C]MNNG, respectively. The amounts of the binding of ENNG were less than those of MNNG, especially in the corporation of the ethyl moiety of ENNG into nucleic acid fractions. In a non-cellular system, the radioactivity of [guanidino-14C]ENNG was incorporated into proteins, preferentially into basic proteins such as cytochrome c, but was not incorporated into nucleic acids. This behavior is similar to that of [guanidino-14C]MNNG, while the amount of binding of the former was about half of that of the latter. The radioactivity of [ethyl-14C]ENNG was also incorporated into basic proteins to almost the same extent as that of [methyl-14C]MNNG. However, the binding of the ethyl moiety of ENNG to nucleic acids was much lower than that of the methyl moiety of MNNG. Horse heart cytochrome c, bovine pancreatic RNase A and regenerating rat liver chromatin had altered their biological activities to various degrees after modification by ENNG or MNNG.     

The Aspergillus nidulans genes chsA and chsD encode chitin synthases which have redundant functions in conidia formation

 We previously isolated three chitin synthase genes (chsA, chsB, and chsC) from Aspergillus nidulans. In the present work, we describe the isolation and characterization of another chitin synthase gene, named chsD, from A. nidulans. Its deduced amino acid sequence shows 56.7% and 55.9% amino acid identity, respectively, with Cal1 of Saccharomyces cerevisiae and Chs3 of Candida albicans. Disruption of chsD caused no defect in cell growth or morphology during the asexual cycle and caused no decrease in chitin content in hyphae. However, double disruption of chsA and chsD caused a remarkable decrease in the efficiency of conidia formation, while double disruption of chsC and chsD caused no defect. Thus it appears that chsA and chsD serve redundant functions in conidia formation.     

Protein A-Mouse Acidic Mammalian Chitinase-V5-His Expressed in Periplasmic Space of Escherichia coli Possesses Chitinase Functions Comparable to CHO-Expressed Protein

Acidic mammalian chitinase (AMCase) has been shown to be associated with asthma in mouse models, allergic inflammation and food processing. Here, we describe an E. coli-expression system that allows for the periplasmic production of active AMCase fused to Protein A at the N-terminus and V5 epitope and (His)₆ tag (V5-His) at the C-terminus (Protein A-AMCase-V5-His) in E. coli. The mouse AMCase cDNA was cloned into the vector pEZZ18, which is an expression vector containing the Staphylococcus Protein A promoter, with the signal sequence and truncated form of Protein A for extracellular expression in E. coli. Most of the Protein A-AMCase-V5-His was present in the periplasmic space with chitinolytic activity, which was measured using a chromogenic substrate, 4-nitrophenyl N,N′-diacetyl-β-D-chitobioside. The Protein A-AMCase-V5-His was purified from periplasmic fractions using an IgG Sepharose column followed by a Ni Sepharose chromatography. The recombinant protein showed a robust peak of activity with a maximum observed activity at pH 2.0, where an optimal temperature was 54°C. When this protein was preincubated between pH 1.0 and pH 11.0 on ice for 1 h, full chitinolytic activity was retained. This protein was also heat-stable till 54°C, both at pH 2.0 and 7.0. The chitinolytic activity of the recombinant AMCase against 4-nitrophenyl N,N′-diacetyl-β-D-chitobioside was comparable to the CHO-expressed AMCase. Furthermore, the recombinant AMCase bound to chitin beads, cleaved colloidal chitin and released mainly N,N′-diacetylchitobiose fragments. Thus, the E. coli-expressed Protein A-mouse AMCase-V5-His fusion protein possesses chitinase functions comparable to the CHO-expressed AMCase. This recombinant protein can be used to elucidate detailed biomedical functions of the mouse AMCase.     

Roles of Poly(3-Hydroxybutyrate) Depolymerase and 3HB-Oligomer Hydrolase in Bacterial PHB Metabolism

Many poly-3-hydroxybutyrate (PHB)-degrading enzymes have been studied. But biological roles of 3HB-oligomer hydrolases (3HBOHs) and how PHB depolymerases (PHBDPs) and 3HBOHs cooperate in PHB metabolism are not fully elucidated. In this study, several PHBDPs and 3HBOHs from three types of bacteria were purified, and their substrate specificity, kinetic properties, and degradation products were investigated. From the results, PHBDP and 3HBOH seemed to play a role in PHB metabolism in three types of bacteria, as follows: (A) In Ralstonia pickettii T1, an extracellular PHBDP degrades extracellular PHB to various-sized 3HB-oligomers, which an extracellular 3HBOH hydrolyzes to 3HB-monomers. (B) In Acidovorax sp. SA1, an extracellular PHBDP hydrolyzes extracellular PHB to small 3HB-oligomers (dimer and trimer), which an intracellular 3HBOH efficiently degrades to 3HB in the cell. (C) In Ralstonia eutropha H16, an intracellular 3HBOH helps in the degradation of intracellular PHB inclusions by PHBDP.