Combined Stimulation of IL-2 and 4-1BB Receptors Augments the Antitumor Activity of E7 DNA Vaccines by Increasing Ag-Specific CTL Responses

Human papillomavirus (HPV) infection is a major cause of cervical cancer. Here, we investigate whether concurrent therapy using HPV E7 DNA vaccines (pE7) plus IL-2 vs. IL-15 cDNA and anti-4-1BB Abs might augment antitumor activity against established tumors. IL-2 cDNA was slightly better than IL-15 cDNA as a pE7 adjuvant. Co-delivery of pE7+IL-2 cDNA increased tumor cure rates from 7% to 27%, whereas co-delivery of pE7+IL-2 cDNA with anti-4-1BB Abs increased tumor cure rates from 27% to 67% and elicited long-term memory responses. This increased activity was concomitant with increased induction of Ag-specific CTL activity and IFN-γ responses, but not with Ag-specific IgG production. Moreover, the combined stimulation of IL-2 and 4-1BB receptors with rIL-2 and anti-4-1BB Abs resulted in enhanced production of IFN-γ from Ag-specific CD8+ T cells. However, this effect was abolished by treatment with anti-IL-2 Abs and 4-1BB-Fc, suggesting that the observed effect was IL-2- and anti-4-1BB Ab-specific. A similar result was also obtained for Ag-specific CTL activity. Thus, these studies demonstrate that combined stimulation through the IL-2 and 4-1BB receptors augments the Ag-specific CD8+ CTL responses induced by pE7, increasing tumor cure rates and long-term antitumor immune memory. These findings may have implications for the design of DNA-based therapeutic vaccines against cancer.     

Cloning of rat brain succinyl-CoA:3-oxoacid CoA-transferase cDNA. Regulation of the mRNA in different rat tissues and during brain development.

3-Oxoacid CoA-transferase, which catalyses the first committed step in the oxidation of ketone bodies, is uniquely regulated in developing rat brain. Changes in 3-oxoacid CoA-transferase activity in rat brain during the postnatal period are due to changes in the relative rate of synthesis of the enzyme. To study the regulation of this enzyme, we identified, with a specific polyclonal rabbit anti-(rat 3-oxoacid CoA-transferase), two positive cDNA clones (approx. 800 bp) in a lambda gtll expression library, constructed from poly(A)+ RNA from brains of 12-day-old rats. One of these clones (lambda CoA3) was subcloned into M13mp18 and subjected to further characterization. Labelled single-stranded probes prepared by primer extension of the M13mp18 recombinant hybridized to a 3.6 kb mRNA. Rat brain mRNA enriched by polysome immunoadsorption for a single protein of size 60 kDa which corresponds to the precursor form of 3-oxoacid CoA-transferase was also found to be similarly enriched for the hybridizable 3.6 kb mRNA complementary to lambda CoA3. Affinity-selected antibody to the lambda CoA3 fusion protein inhibited 3-oxoacid CoA-transferase activity present in rat brain mitochondrial extracts. The 3.6 kb mRNA for 3-oxoacid CoA-transferase was present in relative abundance in rat kidney and heart, to a lesser extent in suckling brain and mammary gland and negligible in the liver. The specific mRNA was also found to be 3-fold more abundant in the brain from 12-day-old rats as compared with 18-day-old foetuses and adult rats, corresponding to the enzyme activity and relative rate of synthesis profile during development. These data suggest that 3-oxoacid CoA-transferase enzyme activity is regulated at a pretranslational level.     

Immunostimulating Effects of Extract of Acanthopanax sessiliflorus

As malfunction/absence of immune cells causes a variety of immunosuppressive disorders and chemical synthetic drugs for curing these diseases have many adverse effects, vigorous studies are being conducted. The Acanthopanax family has been used as traditional medicines for gastric ulcer, diabetes, etc. and culinary materials in East-South Asia. In this study, the immunostimulating properties of A. sessiliflorus were evaluated. A. sessiliflorus increased not only the splenocyte number but also immune-related cytokines such as TNF-α. However, it could not upregulate the expressions of IFN-γ and IL-2. A. sessiliflorus increased the swimming time, and comparison of organ weights relative to body weights for immune-related organs such as the spleen and thymus after a forced swim test showed that it could recover the spleen and thymus weights. It also increased the expression of TNF-α and slightly increased the concentration of IFN-γ but not IL-2. From the results, we concluded that as A. sessiliflorus has not only a host defense effect but also a stress-ameliorating property, further study it will be a promising material of immunostimulating material.     

Extractive lactic acid fermentation in poly (ethyleneimine)-based aqueous two-phase system

The potential of an aqueous two-phase system composed of a polycation, poly(ethyleneimine) (PEI), and an uncharged polymer, (hydroxyethyl) cellulose (HEC), for extractive lactic acid fermentation was tested. Batch fermentation with 20 g/L glucose in two-phase medium using Lactococcus lactis without external pH control resulted in 3-4 times higher amount of lactate and biomass produced as compared to that in a conventional one-phase medium. Lactic acid was preferentially partitioned to the PEI-rich bottom phase. However, the cells which favored the HEC-rich top phase in a fresh two-phase medium were partitioned to a significant extent to the bottom phase after fermentation. Addition of phosphate buffer or pH adjustment to 6.5 after fermentation caused fewer cells to move to the bottom phase. With external pH control, fermentation in normal and two-phase medium showed no marked differences in glucose consumption and lactic acid yield, except that about 1.3 times higher cell density was obtained in the two-phase broth, especially at initial glucose concentrations of 50-100 g/L. Use of higher concentration of phosphate during batch fermentation in the two-phase medium with 50 g/L sugar provided a 15% higher yield of lactic acid, but the growth rate of cells was nearly half of the normal, thus affecting the productivity. Continuous fermentation with twice the normal phosphate concentration resulted in higher cell density, product yield, and productivity in two-phase medium than in monophasic medium. (c) 1996 John Wiley & Sons, Inc.     

Vibrio vulnificus cytolysin induces superoxide anion-initiated apoptotic signaling pathway in human ECV304 cells

Previous studies showed that exposure to Vibrio vulnificus cytolysin (VVC) caused characteristic morphologic changes and dysfunction of vascular structures in lung. VVC showed cytotoxicity for mammalian cells in culture and acted as a vascular permeability factor. In this study, the underlying mechanisms of VVC-induced cytotoxicity was investigated on ECV304 cell, a human vascular endothelial cell line. When cells were exposed to 0.4 hemolytic units (HU) of VVC, consecutive apoptotic events were observed; the elevation of superoxide anion (O (-.)(2)), the release of cytochrome c, the activation of caspase-3, the cleavage of poly(ADP-ribose) polymerase, and the DNA fragmentation. The pretreatment with 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO), O(-.) 2) scavenger, completely abolished O(-.)(2) levels and downstream apoptotic events. Moreover, pretreatment with cyclosporin A (CsA), a mitochondrial permeability transition inhibitor, was capable of attenuating O(-.)(2)-mediated cytochrome c release and caspase-3 activation, and consequent apoptosis. Apoptosis, as demonstrated by oligonucleosomal DNA fragmentation and fluorescence microscopy, was induced 24 h after VVC treatment, which was also prevented by caspase-3 inhibitor, Ac-DEVD-CHO. Caspase-1 inhibitor, Ac-YVAD-CHO, did not protect ECV 304 cells from apoptosis. These results suggest a scenario where VVC-induced apoptosis is triggered by the generation of O(-.)(2), release of cytochrome c from mitochondria, activation of caspase-3, degradation of poly(ADP-ribose) polymerase, and DNA fragmentation. The induction of apoptosis in endothelial cells by VVC may provide a pivotal mechanism for understanding the pathophysiology of septicemia.     

GSK-3 Phosphorylates ��-Catenin and Negatively Regulates Its Stability via Ubiquitination/Proteosome-mediated Proteolysis

δ-Catenin was first identified because of its interaction with presenilin-1, and its aberrant expression has been reported in various human tumors and in patients with Cri-du-Chat syndrome, a form of mental retardation. However, the mechanism whereby δ-catenin is regulated in cells has not been fully elucidated. We investigated the possibility that glycogen-synthase kinase-3 (GSK-3) phosphorylates δ-catenin and thus affects its stability. Initially, we found that the level of δ-catenin was greater and the half-life of δ-catenin was longer in GSK-3β^−/− fibroblasts than those in GSK-3β^+/+ fibroblasts. Furthermore, four different approaches designed to specifically inhibit GSK-3 activity, i.e. GSK-3-specific chemical inhibitors, Wnt-3a conditioned media, small interfering RNAs, and GSK-3α and -3β kinase dead constructs, consistently showed that the levels of endogenous δ-catenin in CWR22Rv-1 prostate carcinoma cells and primary cortical neurons were increased by inhibiting GSK-3 activity. In addition, it was found that both GSK-3α and -3β interact with and phosphorylate δ-catenin. The phosphorylation of ΔC207-δ-catenin (lacking 207 C-terminal residues) and T1078A δ-catenin by GSK-3 was noticeably reduced compared with that of wild type δ-catenin, and the data from liquid chromatography-tandem mass spectrometry analyses suggest that the Thr¹⁰⁷⁸ residue of δ-catenin is one of the GSK-3 phosphorylation sites. Treatment with MG132 or ALLN, specific inhibitors of proteosome-dependent proteolysis, increased δ-catenin levels and caused an accumulation of ubiquitinated δ-catenin. It was also found that GSK-3 triggers the ubiquitination of δ-catenin. These results suggest that GSK-3 interacts with and phosphorylates δ-catenin and thereby negatively affects its stability by enabling its ubiquitination/proteosome-mediated proteolysis.δ-Catenin was first identified as a molecule that interacts with presenilin-1 (PS-1)² by yeast two-hybrid assay (1) and was found to belong to the p120-catenin subfamily of armadillo proteins, which characteristically contain 10 Arm repeats (2). In addition to its interaction with PS-1 and its abundant expression in brain (3, 4), several lines of evidence indicate that δ-catenin may play a pivotal role in cognitive function. First, the hemizygous loss of δ-catenin is known to be closely correlated with Cri-du-Chat syndrome, a severe form of mental retardation in humans (5). Second, severe learning deficits and abnormal synaptic plasticity were found in δ-catenin-deficient mice (6). Moreover, in δ-catenin^−/− mice, paired pulse facilitation (a form of short term plasticity) was found to be reduced, and long term potentiation, which is related to the forming and storage mechanisms of memory, was deficient (7, 8). Third, δ-catenin interacting molecules, such as PSs (1, 9), cadherins (10), S-SCAM (2), and PSD-95 (11), have been shown to play important roles in modulating synaptic plasticity. However, even though the maintenance of an adequate δ-catenin level is known to be critical for normal brain function, few studies have been undertaken to identify the factors that regulate δ-catenin stability in cells. We have previously demonstrated that PS-1 inhibits δ-catenin-induced cellular branching and promotes δ-catenin processing and turnover (12).Because of structural similarities among β-catenin, p120-catenin, and δ-catenin and to their shared binding partners (i.e. PS-1 (1, 9) and cadherins (10)), glycogen-synthase kinase-3 (GSK-3) drew our attention as a potential candidate effector of δ-catenin stability in cells. GSK-3 is a serine/threonine kinase and has two highly homologous forms, GSK-3α and GSK-3β, in mammals (13). Although GSK-3α and GSK-3β have similar structures, they differ in mass (GSK-3α (51 kDa) and GSK-3β (47 kDa) (13)) and to some extent in function (14). GSK-3 is a well established inhibitor of Wnt signaling. Moreover, it is known to phosphorylate β-catenin, which results in its degradation via ubiquitination/proteosome-dependent proteolysis (15). GSK-3 is ubiquitously distributed in the human body, but it is particularly abundant in brain (13), and it is interesting that δ-catenin is also abundant in the nervous system (4) and that GSK-3 participates in the progression of Alzheimer disease (16). The majority of GSK-3 substrates have the consensus sequence (Ser/Thr)-Xaa-Xaa-Xaa-(Ser/Thr) (17). Interestingly, we found that δ-catenin has several putative phosphorylation sites targeted by GSK-3, which suggests that δ-catenin can be regulated by GSK-3 in the same way as β-catenin.In this report, we demonstrate that both GSK-3α and -3β interact with and phosphorylate δ-catenin and that this leads to its subsequent ubiquitination and degradation via proteosome-dependent proteolysis. Our results strongly suggest that GSK-3 is a key regulator of δ-catenin stability in cells.     

Mycobacterial Cytochrome P450 125 (Cyp125) Catalyzes the Terminal Hydroxylation of C27 Steroids

Cyp125 (Rv3545c), a cytochrome P450, is encoded as part of the cholesterol degradation gene cluster conserved among members of the Mycobacterium tuberculosis complex. This enzyme has been implicated in mycobacterial pathogenesis, and a homologue initiates cholesterol catabolism in the soil actinomycete Rhodococcus jostii RHA1. In Mycobacterium bovis BCG, cyp125 was up-regulated 7.1-fold with growth on cholesterol. A cyp125 deletion mutant of BCG did not grow on cholesterol and accumulated 4-cholesten-3-one when incubated in the presence of cholesterol. Wild-type BCG grew on this metabolite. By contrast, a parallel cyp125 deletion mutation of M. tuberculosis H37Rv did not affect growth on cholesterol. Purified Cyp125 from M. tuberculosis, heterologously produced in R. jostii RHA1, bound cholesterol and 4-cholesten-3-one with apparent dissociation constants of 0.20 ± 0.02 μm and 0.27 ± 0.05 μm, respectively. When reconstituted with KshB, the cognate reductase of the ketosteroid 9α-hydroxylase, Cyp125 catalyzed the hydroxylation of these steroids. MS and NMR analyses revealed that hydroxylation occurred at carbon 26 of the steroid side chain, allowing unambiguous classification of Cyp125 as a steroid C26-hydroxylase. This study establishes the catalytic function of Cyp125 and, in identifying an important difference in the catabolic potential of M. bovis and M. tuberculosis, suggests that Cyp125 may have an additional function in pathogenesis.