Citric acid production using immobilized conidia of Aspergillus niger TMB 2022

Conidia of Aspergillus niger TMB 2022 were immobilized in calcium alginate for the production of citric acid. A 1-mL conidia suspension containing ca. 2.32 x 10(8) conidia were entrapped into sodium alginate solution in order to prepare 3% Ca-alginate (w/v) gel bead. Immobilized conidia were inoculated into productive medium containing 14% sucrose, 0.25% (NH(4))(2)CO(3), 0.25% KH(2)PO(4), and 0.025% MgSO(4).7H(2)O with addition of 0.06 mg/L CuSO(4).5H(2)O, 0.25 mg/L ZnCl(2), 1.3 mg/L FeCl(3).6H(2)O, pH 3.8, and incubated at 35 degrees C for 13 days by surface culture to produce 61.53 g/L anhydrous citric acid. Under the same conditions with a batchwise culture, it was found that immobilized conidia could maintain a longer period for citric acid production (31 days): over 70 g/L anhydrous citric acid from runs No. 2-4, with the maximum yield for anhydrous citric acid reaching 77.02 g/L for run No. 2. In contrast, free conidia maintained a shorter acid-producing phase, ca. 17 days; the maximum yield for anhydrous citric acid was 71.07 g/L for run No. 2 but dropped quickly as the run number increased.     

Ornithine decarboxylase prevents tumor necrosis factor alpha-induced apoptosis by decreasing intracellular reactive oxygen species

Ornithine decarboxylase (ODC) plays an essential role in various biological functions, including cell proliferation, differentiation and cell death. However, how it prevents the cell apoptotic mechanism is still unclear. Previous studies have demonstrated that decreasing the activity of ODC by difluoromethylornithine (DFMO), an irreversible inhibitor of ODC, causes the accumulation of intracellular reactive oxygen species (ROS) and cell arrest, thus inducing cell death. These findings might indicate how ODC exerts anti-oxidative and anti-apoptotic effects. In our study, tumor necrosis factor alpha (TNF-) induced apoptosis in HL-60 and Jurkat T cells. The kinetic studies revealed that the TNF- -induced apoptotic process included intracellular ROS generation (as early as 1 h after treatment), the activation of caspase 8 (3 h), the cleavage of Bid (3 h) and the disruption of mitochondrial membrane potential ( _m) (6 h). Furthermore, ROS scavengers, such as glutathione (GSH) and catalase, maintained _m and prevented apoptosis upon treatment. Putrescine and overexpression of ODC had similar effects as ROS scavengers in decreasing intracellular ROS and preventing the disruption of _m and apoptosis. Inhibition of ODC by DFMO in HL-60 cells only could increase ROS generation, but did not disrupt _m or induce apoptosis. However, DFMO enhanced the accumulation of ROS, disruption of _m and apoptosis when cells were treated with TNF- . ODC overexpression avoided the decline of Bcl-2, prevented cytochrome c release from mitochondria and inhibited the activation of caspase 8, 9 and 3. Overexpression of Bcl-2 maintained _m and prevented apoptosis, but could not reduce ROS until four hours after TNF- treatment. According to these data, we suggest that TNF- induces apoptosis mainly by a ROS-dependent, mitochondria-mediated pathway. Furthermore, ODC prevents TNF- -induced apoptosis by decreasing intracellular ROS to avoid Bcl-2 decline, maintain _m, prevent cytochrome c release and deactivate the caspase cascade pathway.     

Identification of the -1 translational frameshift sites using a liquid chromatography-tandem mass spectrometric approach

Translational frameshifting, a ubiquitous mechanism used to produce alternative proteins for different biological purposes, appears in a variety of genes in probably all organisms. In the past, the combinational use of sophisticated expression vectors, specific endopeptidases, and Edman degradation has been the main approach for identification of the translational frameshift sites. Although Edman degradation is highly reliable, it is also time-consuming and costly. In this article, we report a new liquid chromatography-tandem mass spectrometric (LC-MS/MS) approach for identifying the -1 translational frameshift sites. The approach consists of three steps: (i) LC-MS/MS analysis of the protein digests, (ii) primary data analysis using the known mRNA sequence, and (iii) advanced data analysis using a new database containing distinct mRNA sequences with single insertion at particular positions. We first validated our approach by analyzing the previously documented slippery sequence, A4G, from IS3. With this approach, we further determined whether the TTTTTTG (T6G) sequence of IS1372 from Streptomyces lividans had the -1 translational frameshifting potential. The identified amino acid sequence of the transframe peptide indicated that the -1 frameshifting occurred at the T6G motif, as predicted previously. The results on IS3 (A4G) and IS1372 (T6G) suggested that this approach is effective for the translational frameshifting studies.     

Identification of the "missing domain" of the rat copper-transporting ATPase, atp7b: insight into the structural and metal binding characteristics of its N-terminal copper-binding domain

Wilson disease is an autosomal disorder of copper transport caused by mutations in the ATP7B gene encoding a copper-transporting P-type ATPase. The Long Evans Cinnamon (LEC) rat is an established animal model for Wilson disease. We have used structural homology modelling of the N-terminal copper-binding region of the rat atp7b protein (rCBD) to reveal the presence of a domain, the fourth domain (rD4), which was previously thought to be missing from rCBD. Although the CXXC motif is absent from rD4, the overall fold is preserved. Using a wide range of techniques, rCBD is shown to undergo metal-induced secondary and tertiary structural changes similar to WCBD. Competition 65Zn(II)-blot experiments with rCBD demonstrate a binding cooperativity unique to Cu(I). Far-UV circular dichroism (CD) spectra suggest significant secondary structural transformation occurring when 2-3 molar equivalents of Cu(I) is added. Near-UV CD spectra, which indicate tertiary structural transformations, show a proportional decrease in rCBD disulfide bonds upon the incremental addition of Cu(I), and a maximum 5:1 Cu(I) to protein ratio. The similarity of these results to those obtained for the Wilson disease N-terminal copper-binding region (WCBD), which has six copper-binding domains, suggests that the metal-dependent conformational changes observed in both proteins may be largely determined by the protein-protein interactions taking place between the heavy metal-associated (HMA) domains, and remain largely unaffected by the absence of one of the six CXXC copper-binding sites.     

Mechanisms and functional properties of two peptide transporters, AtPTR2 and fPTR2

The Arabidopsis AtPTR2 and fungal fPTR2 genes, which encode H+/dipeptide cotransporters, belong to two different subgroups of the peptide transporter (PTR) (NRT1) family. In this study, the kinetics, substrate specificity, stoichiometry, and voltage dependence of these two transporters expressed in Xenopus oocytes were investigated using the two-microelectrode voltage-clamp method. The results showed that: 1) although AtPTR2 belongs to the same PTR family subgroup as certain H+/nitrate cotransporters, neither AtPTR2 nor fPTR2 exhibited any nitrate transporting activity; 2) AtPTR2 and fPTR2 transported a wide spectrum of dipeptides with apparent affinity constants in the range of 30 microM to 3 mM, the affinity being dependent on the side chain structure of both the N- and C-terminal amino acids; 3) larger maximal currents (Imax) were evoked by positively charged dipeptides in AtPTR2- or fPTR2-injected oocytes; 4) a major difference between AtPTR2 and fPTR2 was that, whereas fPTR2 exhibited low Ala-Asp- transporting activity, AtPTR2 transported Ala-Asp- as efficiently as some of the positively charged dipeptides; 5) kinetic analysis suggested that both fPTR2 and AtPTR2 transported by a random binding, simultaneous transport mechanism. The results also showed that AtPTR2 and fPTR2 were quite distinct from PepT1 and PepT2, two well characterized animal PTR transporters in terms of order of binding of substrate and proton(s), pH sensitivity, and voltage dependence.     

Mutation of a nitrate transporter, AtNRT1:4, results in a reduced petiole nitrate content and altered leaf development

Unlike nitrate uptake of plant roots, less is known at the molecular level about how nitrate is distributed in various plant tissues. In the present study, characterization of the nitrate transporter, AtNRT1:4, revealed a special role of petiole in nitrate homeostasis. Electrophysiological studies using Xenopus oocytes showed that AtNRT1:4 was a low-affinity nitrate transporter. Whole-mount in situ hybridization and RT-PCR demonstrated that AtNRT1:4 was expressed in the leaf petiole. In the wild type, the leaf petiole had low nitrate reductase activity, but a high nitrate content, indicating that it is the storage site for nitrate, whereas, in the atnrt1:4 mutant, the petiole nitrate content was reduced to 50-64% of the wild-type level. Moreover, atnrt1:4 mutant leaves were wider than wild-type leaves. This study revealed a critical role of AtNRT1:4 in regulating leaf nitrate homeostasis, and the deficiency of AtNRT1:4 can alter leaf development.     

Tag1 is an autonomous transposable element that shows somatic excision in both Arabidopsis and tobacco.

Tag1 is a transposable element first identified as an insertion in the CHL1 gene of Arabidopsis. The chl1::Tag1 mutant originated from a plant (ecotype Landsberg erecta) that had been transformed with the maize transposon Activator (Ac), which is distantly related to Tag1. Genomic analysis of untransformed Landsberg erecta plants demonstrated that two identical Tag1 elements are present in the Landsberg erecta genome. To determine what provides transposase function for Tag1 transposition, we examined Tag1 excision in different genetic backgrounds. First, the chl1::Tag1 mutant was backcrossed to untransformed wild-type Arabidopsis plants to remove the Ac element(s) from the genome. F2 progeny that had no Ac elements but still retained Tag1 in the CHL1 gene were identified. Tag1 still excised in these Ac-minus progeny producing CHL1 revertants; therefore, Ac is not required for Tag1 excision. Next, Tag1 was inserted between a cauliflower mosaic virus 35S promoter and a beta-glucuronidase (GUS) marker gene and transformed into tobacco. Transformants showed blue-staining sectors indicative of Tag1 excision. Transgenic tobacco containing a defective Tag1 element, which was constructed in vitro by deleting an internal 1.4-kb EcoRI fragment, did not show blue-staining sectors. We conclude that Tag1 is an autonomous element capable of independent excision. The 35S-GUS::Tag1 construct was then introduced into Arabidopsis. Blue-staining sectors were found in cotyledons, leaves, and roots, showing that Tag1 undergoes somatic excision during vegetative development in its native host.     

Agrobacterium tumefaciens-mediated transformation of Eucalyptus camaldulensis and production of transgenic plants

An efficient system for Agrobacterium-mediated transformation of Eucalyptus camaldulensis and production of transgenic plants was developed. Transformation was accomplished by cocultivation of hypocotyl segments with Agrobacterium tumefaciens containing a binary Ti-plasmid vector harboring chimeric neomycin phosphotransferase and β-glucuronidase (GUS) genes. A modified Gamborg's B5 medium used in this study was effective for both callus induction and regeneration of transgenic shoots. This medium could also effectively maintain the organogenic capability of callus for more than a year. Culturing transgenic shoots in Murashige and Skoog medium supplemented with 0.1 mg ⋅ l^–1 benzylaminopurine prior to root induction in rooting medium markedly increased the rootability of shoots that were recalcitrant to rooting. Histochemical assay revealed the expression of the GUS gene in leaf, stem, and root tissues of transgenic plants. Insertion of the GUS gene in the nuclear genome of transgenic plants was verified by genomic Southern hybridization analysis, further confirming the integration and expression of T-DNA in these plants. Received: 1 August 1997 / Revision received: 11 December 1997 / Accepted: 24 January 1998