Construction of a Pseudozyma antarctica strain without foreign DNA sequences (self-cloning strain) for high yield production of a biodegradable plastic-degrading enzyme

ABSTRACT

The basidiomycetous yeast Pseudozyma antarctica GB-4(0) esterase (PaE) is a promising candidate for accelerating degradation of used biodegradable plastics (BPs). To increase safety and reduce costs associated with the use of PaE, we constructed a self-cloning strain with high-PaE productivity. A Lys12 gene (PaLYS12)-deleted lysine auxotroph strain GB4-(0)-L1 was obtained from GB-4(0) by ultraviolet mutagenesis and nystatin enrichment. Subsequently, the PaE gene (PaCLE1) expression cassette consisting of GB-4(0)-derived PaCLE1, under the control of a xylose-inducible xylanase promoter with PaLYS12, was randomly introduced into the GB4-(0)-L1 genome. A PaE high-producing strain, PGB474, was selected from among the transformants by high throughput double-screening based on its ability to degrade emulsified polybutylene succinate-co-adipate. Quantitative PCR revealed that four copies of the PaE gene expression cassette were introduced into the PGB474 genome. PGB474 produced 2.0 g/L of PaE by xylose-fed-batch cultivation using a 3-L jar fermentor for 72 h.     

Characterization of the stromal protease(s) degrading the cross-linked products of the D1 protein generated by photoinhibition of photosystem II

When photosystem (PS) II-enriched membranes are exposed to strong light, cross-linking of the intrinsic D1 protein with the surrounding polypeptides and degradation of the D1 protein take place. The cross-linking of the D1 protein with the alpha-subunit of cytochrome b(559) is suggested to be an early event of photoinduced damage to the D1 protein (Barbato et al., FEBS Lett. 309 (1992) 165-169). The relationship between the cross-linking and the degradation of the D1 protein, however, is not yet clear. In the present study, we show that the addition of stromal extract from chloroplasts degrades the 41 kDa cross-linked product of D1/cytochrome b(559) alpha-subunit and enhances the degradation of the D1 protein. Incubation of the preilluminated PS II-enriched membranes with the stromal extract at 25 degrees C causes the degradation of the cross-linked product by more than 70%. The activity of the stromal extract showed a pH optimum at 8.0, and was enhanced by the addition of ATP or GTP. Consistent with the nucleotide effect, this stromal activity was eliminated by the preincubation of the stromal extract with apyrase, which hydrolyzes nucleotides. Also, the stromal activity was nearly fully inhibited by a serine-type protease inhibitor, 3,4-dichloroisocoumarin, which suggests participation of a serine-type protease(s).     

Mouse Pitx2 deficiency leads to anomalies of the ventral body wall, heart, extra- and periocular mesoderm and right pulmonary isomerism

Pitx2, a bicoid-related homeobox gene, is involved in Rieger's syndrome and the left-right (L-R) asymmetrical pattern formation in body plan. In order to define the genomic structure and roles of Pitx2, we analyzed the genomic structure and generated Pitx2-deficient mice with the lacZ gene in the homeobox-containing exon of Pitx2. We were able to show that among three isoforms of Pitx2, Pitx2c shows asymmetrical expression whereas Pitx2a, Pitx2b and Pitx2c show symmetrical expression. In Pitx2(-)(/)(-) embryos there was an increase in mesodermal cells in the distal end of the left lateral body wall and an amnion continuous with the lateral body wall thickened in its mesodermal layer. These changes resulted in a failure of ventral body wall closure. In lung and heart in which Pitx2 is expressed asymmetrically, right pulmonary isomerism, atrioventricular canals with prominent swelling, and juxtaposition of the atrium were detected. The hearts failed to develop tricuspid and mitral valves and a common atrioventricular valve forms. Further, dysgenesis of the Pitx2(-)(/)(-) extraocular muscle and thickening of the mesothelial layer of cornea were observed in the ocular system where Pitx2 is expressed symmetrically, and these resulted in enophthalmos. The present study shows that Pitx2 expressed in various sites participates in morphogenesis through three types of actions: the involvement of asymmetric Pitx2 expression in the entire morphogenetic process of L-R asymmetric organs; the involvement of asymmetric Pitx2 expression in the regional morphogenesis of asymmetric organs; and finally the involvement of symmetric Pitx2 expression in the regional morphogenesis of symmetric organs.     

Sequencing of three lambda clones from the genome of alkaliphilic Bacillus sp. strain C-125

The nucleotide sequences of three independent fragments (designated no. 3, 4, and 9; each 15–20 kb in size) of the genome of alkaliphilic Bacillus sp. C-125 cloned in a λ phage vector have been determined. Thirteen putative open reading frames (ORFs) were identified in sequenced fragment no. 3 and 11 ORFs were identified in no. 4. Twenty ORFs were also identified in fragment no. 9. All putative ORFs were analyzed in comparison with the BSORF database and non-redundant protein databases. The functions of 5 ORFs in fragment no. 3 and 3 ORFs in fragment no. 4 were suggested by their significant similarities to known proteins in the database. Among the 20 ORFs in fragment no. 9, the functions of 11 ORFs were similarly suggested. Most of the annotated ORFs in the DNA fragments of the genome of alkaliphilic Bacillus sp. C-125 were conserved in the Bacillus subtilis genome. The organization of ORFs in the genome of strain C-125 was found to differ from the order of genes in the chromosome of B. subtilis, although some gene clusters (ydh, yqi, yer, and yts) were conserved as operon units the same as in B. subtilis. Received: April 17, 1998 / Accepted: June 23, 1998     

Photoperiodic response of serotonin- and galanin-immunoreactive neurons of the paraventricular organ and infundibular nucleus in Japanese quail, Coturnix coturnix japonica

We investigated the photoperiodic response of serotonin- and galanin (GA)- immunoreactive (ir) cells in the paraventricular organ (PVO) and infundibular nucleus (IF) of the Japanese quail and the interaction of these cells with gonadotropin-releasing hormone (GnRH)-ir neurons in the hypothalamus. Serotonin-ir cells were located in series from the PVO to the IF, and were connected with each other. The number of serotonin-ir cells differed significantly between light and dark phases on the short days (SD), but did not differ between light and dark phases on long days (LD). GA-ir cells were also found in the PVO and IF. The number of GA-ir cells under SD conditions was significantly greater than under LD conditions but did not change diurnally. Both serotonin-ir and GA-ir fibers ran along the GnRH-ir cells in the nucleus commissurae pallii. Serotonin-ir and GA-ir fibers were connected with the GnRH-ir fibers in the external layer of the median eminence (ME). We confirmed that GA-ir fibers were closely associated with serotonin-ir neurons in the PVO and IF. GA-ir neurons have at least 2 routes of regulating GnRH neurons directly, and indirectly via the serotonin-ir cells in the PVO and IF.     

Synthesis and biological activity of novel alpha-substituted beta-phenylpropionic acids having pyridin-2-ylphenyl moiety as antihyperglycemic agents

We previously reported the identification of novel oximes having 5-benzyl-2,4-thiazolidinedione with antihyperglycemic activity. We now report the synthesis and biological activity of a novel series of oximes and amides having alpha-substituted-beta-phenylpropionic acids. In this series, we obtained potent PPAR alpha/gamma dual agonist (S)-9d, with which activation of PPAR alpha and PPAR gamma was considerably more potent than that of the reference compounds GW9578 22 and rosiglitazone 3, respectively. This means (S)-9d is of the strongest class of PPAR alpha/gamma dual agonists. In the course of this study, we also obtained 8h, which indicated potent plasma glucose lowering effect in spite of weak PPAR alpha/gamma agonistic activity.     

Serum paraoxonase activity decreases in rheumatoid arthritis

OBJECTIVE: To estimate the alterations of paraoxonase 1 (PON1) and high-density lipoprotein (HDL) in rheumatoid arthritis (RA). DESIGN AND METHODS: We investigated the serum enzyme activity and concentration of PON1 and their relationship with serum lipids, high-density lipoprotein (HDL) parameters, and acute phase reactants of serum amyloid A (SAA) and C-reactive protein (CRP) in patients with RA. RESULTS: Serum paraoxonase (PON) activity was significantly decreased in RA patients (n = 64, 131 +/- 53 micro mol/min/L) compared with healthy subjects (n = 155, 164 +/- 59) despite the absence of any difference in serum lipid levels between the two groups. This decrease of serum PON activity in RA patients was found in every genotype (Q/Q, Q/R, R/R) of PON1 at 192 Q/R. There was a different distribution in PON1 Q/R genotypes between RA patients and healthy subjects, and RA patients exhibited less (44%) positive PON1-Q than did the healthy subjects (66%). In a further investigation of age- and gender-matched subgroups of RA (n = 25) and healthy subjects (n = 25), not only serum PON activity, but also lecithin-cholesterol acyltransferase (LCAT) was found to be significantly decreased in RA patients (125 +/- 61 micro mol/min/L, 63.2 +/- 17.2 nmol/ml/hr/37 degrees C) than in healthy subjects (169 +/- 67, 74.7 +/- 19.5), respectively. PON1 and LCAT as well as HDL constituent apolipoprotein (apo) AI and apo AII, were altered significantly in RA patients. CONCLUSIONS: Acute-phase HDL, which is remodeled structurally and functionally in RA, might be less anti-atherogenic due to the impairment of original HDL function. These alterations of HDL in RA patients may explain in part the reported increase in cardiovascular mortality in patients with RA.     

Molecular taxonomy of dermatophytes and related fungi by chitin synthase 1 (CHS1) gene sequences

In the present study, the nucleotide sequences of the CHS1 gene from dermatophytes and related fungi in the genera Chrysosporium, Epidermophyton, Microsporum and Trichophyton were investigated using molecular methods. About 440-bp genomic DNA fragments of the CHS1 gene from 21 species were amplified by polymerase chain reaction (PCR) and sequenced. The CHS1 nucleotide sequences of these fungi showed more than 83% similarity. The molecular taxonomy of the CHS1 gene sequences revealed that Microsporum was genetically distinct from Chrysosporium and Trichophyton, as classified by morphological characteristics. This revised version was published online in June 2006 with corrections to the Cover Date.     

Establishment of Alb-DsRed2 transgenic rat for liver regeneration research

Because serum albumin is specifically produced by mature hepatocytes, detection system of albumin producing cells could be a valuable tool to visualize liver regeneration or development. We have developed here an albumin enhancer/promoter-driven Alb-DsRed2 Tg rat that expresses DsRed2, having liver-specific reporter gene expression of red fluorescent protein. To study the transdifferentiation of bone marrow cells (BMCs) into albumin producing cells, BMCs from the Alb-DsRed2 Tg rat were injected into rats having acute liver damage caused by 2-acetylaminofluorene plus carbon tetrachloride and chronic liver damage by repeated administration of CCl(4). DsRed2-positive cells were generated in the recipient liver after BMC injection. The number of transdifferentiated DsRed2-positive cells in chronic liver injury model was increased comparing with that in acute injury model. We propose that the Alb-DsRed2 Tg rat is well suited to studying in vivo liver regeneration.