Permanence of single-species stage-structured models

In this paper, we consider population survival by using single-species stage-structured models. As a criterion of population survival, we employ the mathematical notation of permanence. Permanence of stage-structured models has already been studied by Cushing (1998). We generalize his result of permanence, and obtain a condition which guarantees that population survives. The condition is applicable to a wide class of stage-structured models. In particular, we apply our results to the Neubert-Caswell model, which is a typical stage-structured model, and obtain a condition for population survival of the model.The research is partially supported by the Ministry of Education, Science and Culture, Japan, under Grant in Aid for Scientific Research (A) 13304006.     

cDNA cloning and characterization of a secreted luciferase from the luminous Japanese ostracod, Cypridina noctiluca

A secreted luciferase from the marine ostracod, Vargula hilgendorfii, is a useful tool for gene expression assays in living mammalian cells. We have cloned the cDNA of a new secreted luciferase from the ostracod Cypridina noctiluca, which inhabits the coast of Japan. C. noctiluca luciferase consists of 553 amino acid residues with a molecular mass of 61,415 Da, as deduced from the nucleotide sequence. The homologies of nucleotide and amino acid sequences with V. hilgendorfii luciferase are 79.2% and 83.1%, respectively. C. noctiluca luciferase can expressed in and secreted from cultured mammalian cells. The characteristic properties of expressed C. noctiluca luciferase are similar to those of V. hilgendorfii luciferase. However, the activity of C. noctiluca luciferase in culture medium is much higher than that of V. hilgendorfii luciferase, suggesting that C. noctiluca luciferase is a highly potent reporter enzyme for real-time and continuous monitoring of gene expression in living cells.     

GroEL chaperone binding to beetle luciferases and the implications for refolding when co-expressed

The folding of many proteins including luciferase in vivo requires the assistance of molecular chaperone proteins. To understand how a chaperone targets luciferase, we took three luciferases that give different bioluminescence with the same luciferin substrate and with differences in homology. The three luciferase genes, firefly luciferase (FF-Luc) (from Pyrocoelia miyako), and red (RE-Luc) and green (GR-Luc) bioluminescence-emitting luciferases (from Phrixothrix railroad-worms), were expressed in Escherichia coli to produce fusion proteins with predicted molecular masses. Subsequently, we observed that DnaK and GroEL were co-purified along with recombinant luciferase. Although the amount of co-purified DnaK was almost the same compared to FF-Luc, GroEL was 25 and 32 times higher in GR-Luc and RE-Luc respectively. Furthermore, co-expression of GroEL/GroES along with luciferase substantially refolded RE-Luc and GR-Luc compared to FF-Luc.     

Genomic basis for cell-wall diversity in plants. A comparative approach to gene families in rice and Arabidopsis

Monocotyledons and dicotyledons are distinct, not only in their body plans and developmental patterns, but also in the structural features of their cell walls. The recent completion of the rice (Oryza sativa) genomic sequence and publication of the sequence data, together with the completed database of the Arabidopsis thaliana genome, provide the first opportunity to compare the full complement of cell-wall-related genes from the two distinct classes of flowering plants. We made this comparison by exploiting the fact that Arabidopsis and rice have type I and type II walls, respectively, and therefore represent the two extremes in terms of the structural features of plant cell walls. In this review article, we classify all cell-wall-related genes into 32 gene families, and generate their phylogenetic trees. Using these data, we can phylogenetically compare individual genes of particular interest between Arabidopsis and rice. This comparative genome approach shows that the differences in wall architecture in the two plant groups actually mirror the diversity of the individual gene families involved in the cell-wall dynamics of the respective plant species. This study also identifies putative rice orthologs of genes with well-defined functions in Arabidopsis and other plant species.     

Essential 110Cys in active site of membrane-associated prostaglandin E synthase-2

The amino acid sequence of membrane-associated prostaglandin (PG) E synthase-2 (mPGE synthase-2), which has a broad specificity in its thiol requirement for a catalytic activity, has the consensus region from 104Leu to 120Leu found in glutaredoxin and of thioredoxin. The sequence of Cys-x-x-Cys in the consensus region is the active site for thioredoxin and mPGE synthase-2 also has this amino acid sequence (110Cys-x-x-113Cys). The mutation from 110Cys to Ser or the double mutation from 110Cys and 113Cys to Ser caused loss of PGE synthase activity, whereas the single mutation from 113Cys to Ser did not affect the enzyme activity. These results indicate that 110Cys, but not 113Cys, is the essential amino acid in the active site of mPGE synthase-2. 110Cys is an important amino acid in PGE synthase activity and plays the critical role as Cys at the same position in redoxin. Moreover, we found that the reduced form of lipoic acid (dihydrolipoic acid) serves as one of the natural activators of mPGE synthase-2 in the cells.     

Importance of the carbohydrate-binding module of Clostridium stercorarium Xyn10B to xylan hydrolysis

The Clostridium stercorarium xylanase Xyn10B is a modular enzyme comprising two thermostabilizing domains, a family 10 catalytic domain of glycosyl hydrolases, a family 9 carbohydrate-binding module (CBM), and two S-layer homologous (SLH) domains [Biosci. Biotechnol. Biochem., 63, 1596-1604 (1999)]. To investigate the role of this CBM, we constructed two derivatives of Xyn10B and compared their hydrolytic activity toward xylan and some preparations of plant cell walls; Xyn10BdeltaCBM consists of a catalytic domain only, and Xyn10B-CBM comprises a catalytic domain and a CBM. Xyn10B-CBM bound to various insoluble polysaccharides including Avicel, acid-swollen cellulose, ball-milled chitin, Sephadex G-25, and amylose-resin. A cellulose binding assay in the presence of soluble saccharides suggested that the CBM of Xyn10B had an affinity for even monosaccharides such as glucose, galactose, xylose, mannose and ribose. Removal of the CBM from the enzyme negated its cellulose- and xylan-binding abilities and severely reduced its enzyme activity toward insoluble xylan and plant cell walls but not soluble xylan. These findings clearly indicated that the CBM of Xyn10B is important in the hydrolysis of insoluble xylan. This is the first report of a family 9 CBM with an affinity for insoluble xylan in addition to crystalline cellulose and the ability to increase hydrolytic activity toward insoluble xylan.     

Inhibition of EBV-induced lymphoproliferation by CD4(+) T cells specific for an MHC class II promiscuous epitope

Posttransplant lymphoproliferative disorder (PTLD) and B cell lymphomas induced by EBV continue to be a major life-threatening complication in transplant patients. The establishment and enhancement of T cell immunity to EBV before transplantation and immunosuppressive therapy could help diminish these complications, but the lack of an effective vaccine has limited this prophylactic approach. We describe here the identification of a peptide epitope from the EBV EBNA2 Ag that is capable of inducing in vitro CD4(+) T cell responses that inhibit the EBV-mediated B lymphocyte proliferation associated with PTLD. Most significantly, T cell responses to the EBNA2 epitope were found to be restricted by numerous MHC class II alleles (DR1, DR7, DR16, DR52, DQ2, and DQ7), indicating that this peptide is highly promiscuous and would be recognized by a large proportion (>50%) of the general population. These results are relevant for the design of a simple, inexpensive and widely applicable peptide-based vaccine to prevent PTLD in solid organ transplant patients.     

Molecular cloning, characterization, and expression analysis of the xynF3 gene from Aspergillus oryzae

The gene encoding xylanase F3 (xynF3) was isolated from a genomic library of Aspergillus oryzae KBN616, used for making shoyu koji. The structural part of xynF3 was found to be 1468 bp. The nucleotide sequence of cDNA amplified by RT-PCR showed that the open reading frame of xynF3 was interrupted by ten short introns and encoded 323 amino acids. Direct N-terminal amino acid sequencing showed that the precursor of XynF3 had a signal peptide of 22 amino acids. The predicted amino acid sequence of XynF3 has strong similarity to other family 10 xylanases from fungi. The xynF3 gene was successfully overexpressed in A. oryzae and the XynF3 was purified. The molecular mass of XynF3 estimated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 32,000. This was almost the same as the molecular mass of 32,437 calculated from the deduced amino acid sequence. The purified XynF3 showed an optimum activity at pH 5.0 and 58 degrees C. It had a Km of 6.5 mg/ml and a Vmax of 435 micromol x min(-1) x mg(-1) when birch wood xylan was used as a substrate. Expression of the xynF3 gene was analyzed using an Escherichia coli beta-glucuronidase gene as a reporter. The result indicated that xynF3 is expressed in the medium containing wheat bran as a carbon source.     

Molecular breeding of transgenic rice expressing a xylanase domain of the<Emphasis Type="Italic"> xynA</Emphasis> gene from<Emphasis Type="Italic"> Clostridium thermocellum</Emphasis>

The gene encoding the catalytic domain of thermostable xylanase from Clostridium thermocellum F1 was expressed in rice plants under the control of a constitutive promoter. The gene encoding Xylanase A was modified to encode the catalytic domain of family 11 xylanase without the signal sequence (xynA1), and was introduced into rice plants and expressed under the control of a modified cauliflower mosaic virus 35S promoter. Zymogram analysis indicated that the recombinant xylanase was produced in rice plants. The xynA1 gene was stably expressed in rice straw and seed grains. No phenotypic effect of xylanase expression was noted. The enzyme was detected in the desiccated grain. High levels of enzyme activity were maintained in the cell-free extract during incubation at 60 degrees C for 24 h. The results indicated that high levels of xylanase can be produced in rice plants.