Separate control of rep and cap expression using mutant and wild-type LoxP sequences and improved packaging system for adeno-associated virus vector production

Adeno-associated virus (AAV) vectors are a practical choice for gene transfer, and demand for them is increasing. To cope with the necessity in the near future, we have developed a number of approaches to establish packaging cell lines for the production of AAV vectors. In our previous study, a highly regulated expression of large Rep proteins was obtained by using the Cre-loxP switching system. Therefore, in the present study, to regulate Cap expression as well, we developed an inducible expression system for both Rep and Cap proteins by using an additional set of mutant loxP sequences. The mutants possess two base alterations in the spacer region of loxP and recombine specifically with the same counterpart in the presence of Cre. By using two separate plasmids, one with mutant and the other with wild-type loxP sequences, the expression of two different proteins can be induced simultaneously by Cre recombinase. When the LacZencoding plasmid vector was used as a packaging model, a significant packaging titer of 2.1 × 10¹⁰ genome copies per 10-cm dish was obtained. These results indicate the importance of controlling Cap expression, in addition to Rep, to achieve an optimum production rate for AAV vectors.     

A new cell-permeable peptide allows successful allogeneic islet transplantation in mice

Calcineurin inhibitors such as cyclosporine A and FK506 have been used for transplant therapy and treatment of autoimmune diseases. However, the inhibition of calcineurin outside the immune system has a number of side effects, including hyperglycemia. In the search for safer drugs, we developed a cell-permeable inhibitor of NFAT (nuclear factor of activated T cells) using the polyarginine peptide delivery system. This peptide provided immunosuppression for fully mismatched islet allografts in mice. In addition, it did not affect insulin secretion, whereas FK506 caused a dose-dependent decrease in insulin secretion. Cell-permeable peptides can thus provide a new strategy for drug development and may eventually be useful clinically.     

C-terminal periplasmic domain of Escherichia coli quinoprotein glucose dehydrogenase transfers electrons to ubiquinone

Membrane-bound quinoprotein glucose dehydrogenase (GDH) in Escherichia coli donates electrons directly to ubiquinone during the oxidation of d-glucose as a substrate, and these electrons are subsequently transferred to ubiquinol oxidase in the respiratory chain. To determine whether the specific ubiquinone-reacting site of GDH resides in the N-terminal transmembrane domain or in the large C-terminal periplasmic catalytic domain (cGDH), we constructed a fusion protein between the signal sequence of beta-lactamase and cGDH. This truncated GDH was found to complement a GDH gene-disrupted strain in vivo. The signal sequence of the fused protein was shown to be cleaved off, and the remaining cGDH was shown to be recovered in the membrane fraction, suggesting that cGDH has a membrane-interacting site that is responsible for binding to membrane, like peripheral proteins. Kinetic analysis and reconstitution experiments revealed that cGDH has ubiquinone reductase activity nearly equivalent to that of the wild-type GDH. Thus, it is likely that the C-terminal periplasmic domain of GDH possesses a ubiquinone-reacting site and transfers electrons directly to ubiquinone.     

Functions of amino acid residues in the active site of Escherichia coli pyrroloquinoline quinone-containing quinoprotein glucose dehydrogenase

Several mutants of quinoprotein glucose dehydrogenase (GDH) in Escherichia coli, located around its cofactor pyrroloquinoline quinone (PQQ), were constructed by site-specific mutagenesis and characterized by enzymatic and kinetic analyses. Of these, critical mutants were further characterized after purification or by different amino acid substitutions. H262A mutant showed reduced affinities both for glucose and PQQ without significant effect on glucose oxidase activity, indicating that His-262 occurs very close to PQQ and glucose, but is not the electron acceptor from PQQH(2). W404A and W404F showed pronounced reductions of affinity for PQQ, and the latter rather than the former had equivalent glucose oxidase activity to the wild type, suggesting that Trp-404 may be a support for PQQ and important for the positioning of PQQ. D466N, D466E, and K493A showed very low glucose oxidase activities without influence on the affinity for PQQ. Judging from the enzyme activities of D466E and K493A, as well as their absorption spectra of PQQ during glucose oxidation, we conclude that Asp-466 initiates glucose oxidation reaction by abstraction of a proton from glucose and Lys-493 is involved in electron transfer from PQQH(2).     

Regulated expression of endothelial cell-derived lipase

A lipoprotein lipase-like gene was recently cloned from endothelial cells. In vitro functional experiments have suggested that this endothelial-derived lipase (EDL) has phospholipase activity, and preliminary in vivo studies have suggested a role in the regulation of high-density lipoprotein metabolism. To investigate local control of lipase activity and lipid metabolism in the blood vessel wall, we have examined the regulation of EDL expression in cultured human umbilical vein and coronary artery endothelial cells. EDL mRNA levels were upregulated in both cell types by inflammatory cytokines implicated in vascular disease etiology, including TNF-alpha and IL-1beta. In addition, both fluid shear stress and cyclic stretch were found to increase the EDL mRNA levels in these cultured cells. This highly regulated expression of EDL in vascular endothelial cells suggests that this recently identified lipase is intricately involved in modulating vessel wall lipid metabolism and may play a role in vascular diseases such as atherosclerosis.     

CYP2D6 polymorphism and the presence of anti-LKM-1 in patients with chronic hepatitis C

Anti-LKM-1 autoantibodies are directed mostly at cytochrome P450 2D6 (CYP2D6) autoantigen, whose activity ranges from "complete deficiency" to "extensive metabolism" due to genetic polymorphism. We aimed to find any relevance of CYP2D6 alleles to the presence/absence of anti-LKM-1 in Japanese patients with chronic hepatitis C. The frequency of an extensive metabolizer-type allele (CYP2D6*1) in anti-LKM-1-positive patients was higher than that in anti-LKM-1-negative patients (0.800 vs 0.431; P = 0.0035), while the CYP2D6*10 allele with moderately reduced activity was less frequent in the former than the latter (0.050 vs 0.389; P = 0.0069). Moreover, the rate of homozygosity for CYP2D6*1 showed a striking difference between the two groups (70% vs 19%; P = 0.0021). These findings suggest that a genetic predisposition to produce the enzyme CYP2D6 of extensive metabolizer-type is associated with the induction of anti-LKM-1 in chronic hepatitis C patients.     

Dynamic aspects of the structured cell population in a swarming colony of Proteus mirabilis

Proteus mirabilis forms a concentric-ring colony by undergoing periodic swarming. A colony in the process of such synchronized expansion was examined for its internal population structure. In alternating phases, i.e., swarming (active migration) and consolidation (growth without colony perimeter expansion), phase-specific distribution of cells differing in length, in situ mobility, and migration ability on an agar medium were recognized. In the consolidation phase, the distribution of mobile cells was restricted to the inner part of a new ring and a previous terrace. Cells composing the outer part of the ring were immobile in spite of their ordinary swimming ability in a viscous solution. A sectorial cell population having such an internal structure was replica printed on fresh agar medium. After printing, a transplant which was in the swarming phase continued its ongoing swarming while a transplanted consolidation front continued its scheduled consolidation. This shows that cessation of migration during the consolidation phase was not due to substances present in the underlying agar medium. The ongoing swarming schedule was modifiable by separative cutting of the swarming front or disruption of the ring pattern by random mixing of the pattern-forming cell population. The structured cell population seemed to play a role in characteristic colony growth. However, separation of a narrow consolidation front from a backward area did not induce disturbance in the ongoing swarming schedule. Thus, cells at the frontal part of consolidation area were independent of the internal cell population and destined to exert consolidation and swarming with the ongoing ordinary schedule.     

Requirement of divalent galactoside-binding activity of ecalectin/galectin-9 for eosinophil chemoattraction

We have previously isolated and cloned a novel eosinophil chemoattractant (ECA) from a human T-cell-derived expression library. This ECA, termed ecalectin, is a variant of human galectin-9, a member of a beta-galactoside binding animal lectin family, which contains two conserved carbohydrate recognition domains (CRDs). In the present study, we addressed whether carbohydrate binding activity is required for the ECA activity of ecalectin and whether both CRDs are essential for this activity. Recombinant full-length wild-type ecalectin (ecalectin-WT) and N-terminal and C-terminal CRD (ecalectin-NT and -CT, respectively) were generated. All of these recombinant proteins exhibited affinity for lactose, a property shared by galectins, but ecalectin-WT exhibited substantially higher hemagglutination activities than ecalectin-NT and -CT. Furthermore, ecalectin-WT showed over 100-fold higher ECA activity than ecalectin-NT and -CT; combination of recombinant domain fragments did not reconstitute the ECA and hemagglutination activities of the full-length protein. ECA activity of ecalectin-WT was inhibited by lactose in a dose-dependent manner. Site-directed mutation of positions Arg(65) of ecalectin-NT and Arg(239) of ecalectin-CT to an aspartic acid residue resulted in the loss of both lactose-binding and ECA activities. We conclude that divalent galactoside-binding activity is required for eosinophil chemoattraction by ecalectin.     

Ligation of HLA-DR molecules on B cells induces enhanced expression of IgM heavy chain genes in association with Syk activation

Signals transmitted by class II major histocompatibility complex are important regarding cell function related to antigen presentation. We examined effects of DR-mediated signaling on Ig production from B cells. Cross-linking HLA-DR molecules on B cells by solid-phase anti-HLA-DR monoclonal antibodies, led to an increased production of IgM, without proliferation or apoptosis. This event was accompanied by an enhanced expression of both membrane- and secretory-type IgM heavy chain mRNA. When peptide-pulsed B cells were co-incubated with an HLA-DR-restricted T cell clone treated by the protein synthesis inhibitor emetine, peptide-induced de novo expression of lymphokines and cell-surface molecules on T cells can be neglected. CD40-CD154 interaction was not involved in IgM enhancement, in such a system. The protein-tyrosine kinase inhibitors and the Syk inhibitor piceatannol, but not the Src inhibitor PP2 had a marked inhibitory effect on IgM secretion. Furthermore, ligation of HLA-DR on B cells using the F(ab')2 fragment of anti-DR monoclonal antibody, enhanced Syk activity. Our data suggest that HLA-DR on B cells not only present antigenic peptides to T cells, but also up-regulate IgM production, in association with Syk activation and without the involvement of Src kinases, hence the possible physiological relevance of Src-independent Syk activation.