Enzymatic synthesis of 1,3,6,8-tetrahydroxynaphthalene solely from malonyl coenzyme A by a fungal iterative type I polyketide synthase PKS1

The Colletotrichum lagenarium PKS1 gene encoding iterative type I polyketide synthase of 1,3,6,8-tetrahydroxynaphthalene (T4HN) was overexpressed in Aspergillus oryzae. SDS-PAGE analysis of the cell-free extract prepared from the transformant showed an intense band of 230000 which corresponded to the molecular weight of the deduced PKS1 protein. By using this cell-free extract, in vitro synthesis of T4HN was successfully confirmed as the first example of the fungal multi-aromatic ring polyketide synthase activity ever detected. To identify the starter unit for T4HN synthesis, (14)C-labeled acetyl CoA and/or (14)C-labeled malonyl CoA were used as substrates for T4HN synthase reaction. Observed was the incorporation of (14)C label into T4HN solely from malonyl CoA even in the absence of acetyl CoA and not from acetyl CoA. This in vitro result unambiguously identified that malonyl CoA serves as the starter as well as extender units in the formation of T4HN by fungal polyketide synthase PKS1.     

Apoptotic wing degeneration and formation of an altruism-regulating glandular appendage (gemma) in the ponerine ant <Emphasis Type="Italic">Diacamma</Emphasis> sp. from Japan (Hymenoptera,Formicidae, Ponerinae)

We here show an example of morphological novelties, which have evolved from insect wings into the specific structures controlling social behaviour in an ant species. Most ant colonies consist of winged queen(s) and wingless workers. In the queenless ponerine ant Diacamma sp. from Japan, however, all female workers have a pair of small thoracic appendages, called gemmae, which are homologous to the forewings and acts as an organ regulating altruism expression. Most workers, whose gemmae are clipped off by other colony members, become nonreproductive helpers, while only a single individual with complete gemmae becomes functionally reproductive. We examined histologically the development of gemmae, and compared it with that of functional wings in males. Female larvae had well-developed wing discs for both fore- and hindwings. At pupation, however, the wing discs started to evaginate and later degenerate. The hindwing discs completely degenerated, while the degeneration of forewing discs was incomplete, leading to the formation of gemmae. The degeneration process involved apoptotic cell death as confirmed by TUNEL assay. In addition, glandular cells differentiated from the epithelial cells of the forewing buds after completion of pupation. The mechanism of developmental transition from wing to gemma can be regarded as an evolutionary gain of new function, which can be seen in insect appendages and vertebrate limbs.Edited by P. Simpson     

Effects of acid exposure on the conformation, stability, and aggregation of monoclonal antibodies

Exposure of antibodies to low pH is often unavoidable for purification and viral clearance. The conformation and stability of two humanized monoclonal antibodies (hIgG4-A and -B) directed against different antigens and a mouse monoclonal antibody (mIgG1) in 0.1M citrate at acidic pH were studied using circular dichroism (CD), differential scanning calorimetry (DSC), and sedimentation velocity. Near- and far-UV CD spectra showed that exposure of these antibodies to pH 2.7-3.9 induced only limited conformational changes, although the changes were greater at the lower pH. However, the acid conformation is far from unfolded or so-called molten globule structure. Incubation of hIgG4-A at pH 2.7 and 3.5 at 4 degrees C over the course of 24 h caused little change in the near-UV CD spectra, indicating that the acid conformation is stable. Sedimentation velocity showed that the hIgG4-A is largely monomeric at pH 2.7 and 3.5 as well as at pH 6.0. No time-dependent changes in sedimentation profile occurred upon incubation at these low pHs, consistent with the conformational stability observed by CD. The sedimentation coefficient of the monomer at pH 2.7 or 3.5 again suggested that no gross conformational changes occur at these pHs. DSC analysis of the antibodies showed thermal unfolding at pH 2.7-3.9 as well as at pH 6.0, but with decreased melting temperatures at the lower pH. These results are consistent with the view that the antibodies undergo limited conformational change, and that incubation at 4 degrees C at low pH results in no time-dependent conformational changes. Titration of hIgG4-A from pH 3.5 to 6.0 resulted in recovery of native monomeric proteins whose CD and DSC profiles resembled those of the original sample. However, titration from pH 2.7 resulted in lower recovery of monomeric antibody, indicating that the greater conformational changes observed at this pH cannot be fully reversed to the native structure by a simple pH titration.     

Mitochondria-specific RNA-modifying enzymes responsible for the biosynthesis of the wobble base in mitochondrial tRNAs. Implications for the molecular pathogenesis of human mitochondrial diseases

Human mitochondrial (mt) tRNA(Lys) has a taurine-containing modified uridine, 5-taurinomethyl-2-thiouridine (taum5s2U), at its anticodon wobble position. We previously found that the mt tRNA(Lys), carrying the A8344G mutation from cells of patients with myoclonus epilepsy associated with ragged-red fibers (MERRF), lacks the taum5s2U modification. Here we describe the identification and characterization of a tRNA-modifying enzyme MTU1 (mitochondrial tRNA-specific 2-thiouridylase 1) that is responsible for the 2-thiolation of the wobble position in human and yeast mt tRNAs. Disruption of the yeast MTU1 gene eliminated the 2-thio modification of mt tRNAs and impaired mitochondrial protein synthesis, which led to reduced respiratory activity. Furthermore, when MTO1 or MSS1, which are responsible for the C5 substituent of the modified uridine, was disrupted along with MTU1, a much more severe reduction in mitochondrial activity was observed. Thus, the C5 and 2-thio modifications act synergistically in promoting efficient cognate codon decoding. Partial inactivation of MTU1 in HeLa cells by small interference RNA also reduced their oxygen consumption and resulted in mitochondria with defective membrane potentials, which are similar phenotypic features observed in MERRF.     

Wobble modification deficiency in mutant tRNAs in patients with mitochondrial diseases

Point mutations in mitochondrial (mt) tRNA genes are associated with a variety of human mitochondrial diseases. We have shown previously that mt tRNA(Leu(UUR)) with a MELAS A3243G mutation and mt tRNA(Lys) with a MERRF A8344G mutation derived from HeLa background cybrid cells are deficient in normal taurine-containing modifications [taum(5)(s(2))U; 5-taurinomethyl-(2-thio)uridine] at the anticodon wobble position in both cases. The wobble modification deficiency results in defective translation. We report here wobble modification deficiencies of mutant mt tRNAs from cybrid cells with different nuclear backgrounds, as well as from patient tissues. These findings demonstrate the generality of the wobble modification deficiency in mutant tRNAs in MELAS and MERRF.     

Cloning, sequencing, and expression of the gene encoding an intracellular beta-D-xylosidase from Streptomyces thermoviolaceus OPC-520

The intracellular beta-xylosidase was induced when Streptomyces thermoviolaceus OPC-520 was grown at 50 degrees C in a minimal medium containing xylan or xylooligosaccharides. The 82-kDa protein with beta-xylosidase activity was partially purified and its N-terminal amino acid sequence was analyzed. The gene encoding the enzyme was cloned, sequenced, and expressed in Escherichia coli. The bxlA gene consists of a 2,100-bp open reading frame encoding 770 amino acids. The deduced amino acid sequence of the bxlA gene product had significant similarity with beta-xylosidases classified into family 3 of glycosyl hydrolases. The bxlA gene was expressed in E. coli, and the recombinant protein was purified to homogeneity. The enzyme was a monomer with a molecular mass of 82 kDa. The purified enzyme showed hydrolytic activity towards only p-nitrophenyl-beta-D-xylopyranoside among the synthetic glycosides tested. Thin-layer chromatography analysis showed that the enzyme is an exo-type enzyme that hydrolyze xylooligosaccharides, but had no activity toward xylan. High activity against pNPX occurred in the pH range 6.0-7.0 and temperature range 40-50 degrees C.     

Tooth regeneration from newly established cell lines from a molar tooth germ epithelium

In order to investigate tooth development, several cell lines of the dental epithelium and ectomesenchyme have been established. However, no attempt has been reported to regenerate teeth with cell lines. Here, we have established several clonal cell lines of the dental epithelium from a p53-deficient fetal mouse. They expressed specific markers of the dental epithelium such as ameloblastin and amelogenin. A new method has been developed to bioengineer tooth germs with dental epithelial and mesenchymal cells. Reconstructed tooth germs with cell lines and fetal mesenchymal cells were implanted under kidney capsule. The germs regenerated teeth with well-calcified structures as seen in natural tooth. Germs without the cell lines developed bone. This is the first success to regenerate teeth with dental epithelial cell lines. They are useful models in vitro for investigation of mechanisms in morphogenesis and of cell lineage in differentiation, and for clinical application for tooth regeneration.     

Studies on the ɛ-Lysine Acylase

?-Lysine acylase of Achromobacter pestifer EA was purified by fractionations with ammonium sulfate and acetone, and by vertical zone electrophoresis. As a result, this bacterial ?-lysine acylase was obtained as an electrophoretically homogeneous protein, specific activity of which is the highest among ?-lysine acylases ever reported.     

Recent developments in yeast cell surface display toward extended applications in biotechnology

Yeasts are promising hosts for industrial bio-refinery applications. In yeast cell surface displays, functional proteins, such as cellulases or lipases, are genetically fused to an anchor protein and expressed on the cell surface. Saccharomyces cerevisiae is the most commonly used yeast for cell surface display. Engineered yeasts have been utilized for a variety of applications, such as bioethanol production, chemicals synthesis, adsorption of environmental pollutants, and protein evolution. Here, we summarize recent developments in yeast cell surface display techniques for bio-refinery applications, including methods using hosts such as Pichia pastoris, Yarrowia lipolytica, and S. cerevisiae, focusing on the characteristics of anchor proteins and applications.     

Cloning and molecular characterization of a cubilin-related serine proteinase from the hard tick Haemaphysalis longicornis

Serine proteinases are one of the largest proteolytic families of enzymes, and have diverse cellular activities in mammalian tissues. We report here the cloning and molecular characterization of a cDNA encoding the serine proteinase of the hard tick Haemaphysalis longicornis (HlSP). The HlSP cDNA is 1570 bp long and the deduced precursor protein consists of 464 amino acids with a predicted molecular mass of 50.4 kDa and a pI of 8.2. The preprotein, consisting of 443 amino acids, was predicted to include a complement C1r/C1s, Uegf, and bone morphogenic protein-1 domain, a low-density lipoprotein receptor class A domain, and a catalytic domain. HlSP sequence analysis showed high similarity to serine proteinases reported from arthropods and vertebrate animal species. Two-dimensional immunoblot analysis revealed endogenous HlSP in adult tick extracts at 50 kDa. Endogenous HlSP was also expressed in all lifecycle stages of H. longicornis. Immunohistochemical studies detected the endogenous enzyme in the midgut epithelial cells of an adult tick. The Escherichia coli-expressed recombinant HlSP was demonstrated to degrade bovine serum albumin and hydrolyze the substrate Bz-L-Arg-pNA at the rate of 30.2 micromol/min/mg protein. Further, HlSP expression was up-regulated during a blood-feeding process, indicating its involvement in the digestion of host blood components.