The promoter of Milk vetch dwarf virus component 8 confers effective gene expression in both dicot and monocot plants

The activity of a predicted promoter, PMC8, from Milk vetch dwarf virus was evaluated by comparing it with the cauliflower mosaic virus 35S RNA promoter (P35S) and PNCR, a promoter from Soybean chlorotic mottle virus. When the GUS fusion gene was introduced into tobacco, PMC8 showed a similar expression profile to P35S but with a more intense expression in proliferating tissues. The usefulness of PMC8 was confirmed by driving NPTII for selection of kanamycin-resistant tobacco plants with improved transformation efficiency. PMC8 was also effective in transgenic rice plants. Thus, PMC8 is useful as an alternative to P35S in both dicotyledonous and monocotyledonous plants, especially for gene expression in proliferating tissues.     

Implication of Ia-positive bone marrow interstitial stem cells in the induction of unresponsiveness to canine renal allografts

The removal from stored autologous host bone marrow of a monocytoid cell population by exposure to methylprednisolone is associated with successful introduction of unresponsiveness to renal allografts in irradiated recipients reconstituted with such treated marrow. The eliminated cells are a prominent component of the canine long bone marrow interstitium and share a number of important properties with dendritic cells (DC), including size and shape; poor or nonadherence to plastic or glass surfaces; negative staining for neutral esterase, acid phosphatase, or peroxidase; nonphagocytic; Ia positive, but negative for IgG or IgM; ability to act as accessory cells in augmenting the intensity of allogeneic mixed-lymphocyte reactions. Both cell types are of bone marrow origin and are susceptible to steroids in vitro. The results suggest that the bone marrow interstitial cells identified in the course of this study may be enriched with populations of canine dendritic cell precursors and dendritic cells at various stages of differentiation. The detection of a receptor site for Helix promatia on the surface of such cells may be of usefulness in their further characterization and in the analysis of their precise role in the modulation of allogeneic unresponsiveness.     

Tightly Regulated Expression of Autographa californica Multicapsid Nucleopolyhedrovirus Immediate Early Genes Emerges from Their Interactions and Possible Collective Behaviors

To infect their hosts, DNA viruses must successfully initiate the expression of viral genes that control subsequent viral gene expression and manipulate the host environment. Viral genes that are immediately expressed upon infection play critical roles in the early infection process. In this study, we investigated the expression and regulation of five canonical regulatory immediate-early (IE) genes of Autographa californica multicapsid nucleopolyhedrovirus: ie0, ie1, ie2, me53, and pe38. A systematic transient gene-expression analysis revealed that these IE genes are generally transactivators, suggesting the existence of a highly interactive regulatory network. A genetic analysis using gene knockout viruses demonstrated that the expression of these IE genes was tolerant to the single deletions of activator IE genes in the early stage of infection. A network graph analysis on the regulatory relationships observed in the transient expression analysis suggested that the robustness of IE gene expression is due to the organization of the IE gene regulatory network and how each IE gene is activated. However, some regulatory relationships detected by the genetic analysis were contradictory to those observed in the transient expression analysis, especially for IE0-mediated regulation. Statistical modeling, combined with genetic analysis using knockout alleles for ie0 and ie1, showed that the repressor function of ie0 was due to the interaction between ie0 and ie1, not ie0 itself. Taken together, these systematic approaches provided insight into the topology and nature of the IE gene regulatory network.     

Bovine Pituitary Membrane Glycoproteins Contain β-N-Acetylgalactosaminylated N-Linked Sugar Chains

Abstract: Bovine pituitary glycoprotein hormones contain unique N-linked sugar chains with GalNAcβ1 → 4GlcNAc structure in their outer chain moieties. In the present study, whether bovine pituitary membrane glycoproteins contain the sugar chains with the disaccharide structure was investigated. Western blot analysis of the membrane glycoproteins using Wistaria floribunda agglutinin (WFA), which binds oligosaccharides terminating with β- N -acetylgalactosamine residue(s), showed that most protein bands detected with Coomassie Brilliant Blue staining bind to WFA. However, no WFA binding was observed for the bands after treatment of the blotted filter with jack bean β- N -acetylhexosaminidase or N -Glycanase. The WFA-positive bands were also detected in membrane glycoprotein samples from bovine cerebrum, cerebellum, and medulla oblongata, although their expression levels were low. Structural analysis of the oligosaccharides released by hydrazinolysis from the pituitary membrane glycoproteins by serial lectin column chromatography and sequential exoglycosidase digestion revealed that the major oligosaccharides, which bound to a WFA-agarose column, are of biantennary complex type with one and two GalNAcβ1 → 4GlcNAc groups in their outer chain moieties. These results indicate that the β- N -acetylgalactosaminylation is not unique to the glycoprotein hormones but occurs to most bovine pituitary glycoproteins.     

Suppression of experimental allergic encephalomyelitis (EAE) with liposome-encapsulated protease inhibitor: Therapy through the blood-brain barrier

Experimental allergic encephalomyelitis (EAE) is an experimentally induced autoallergic demyelinating disease which is caused by immunization with a particular neuroantigen, such as myelin basic protein (MBP). Results have suggested that protease inhibitors might be useful therapeutically. Leupeptin (acetyl-l-leucyl-l-leucyl-argininal), a protease inhibitor of tripeptide nature, was effective in suppressing EAE in guinea pigs, when administered in a form of liposomes consisting of egg lecithin, cholesterol and sulfatide. The drug seemed to be transported into the central nervous system (CNS) tissues across the blood-brain barrier with the aid of a particular type of liposomes as vehicle. Some outbred Hartley guinea pigs completely recovered from distinct symptoms of EAE, such as loss of weight, paralysis, incontinence and/or diarrhea, when treated i.p. every day with lecithin-cholesterol-sulfatide (molar ratio, 451) reverse-phase evaporation vesicles-encapsulated leupeptin (REV-Leu) from day 6 after sesitization with 30 g of bovine MBP. Scarcely any typical histopathological changes of EAE were found in the CNS of most survivors treated with REV-Leu.Special Issue dedicated to Dr. Elizabeth Roboz-Einstein.     

Nitrated and oxidized products of a single tryptophan residue in human Cu,Zn-superoxide dismutase treated with either peroxynitrite-carbon dioxide or myeloperoxidase-hydrogen peroxide-nitrite

We reported previously that a single tryptophan residue, Trp32, in human Cu,Zn-superoxide dismutase is specifically modified by peroxynitrite-CO2 [Yamakura et al. (2001) Biochim. Biophys. Acta 1548, 38-46]. In this study, we modified Cu,Zn-superoxide dismutase by using a combination of myeloperoxidase, hydrogen peroxide, and nitrite. The modified enzyme showed no loss of copper and zinc, and 15% less enzymatic activity. Trp32 was the only significant amino acid lost. After trypsin digestion of the modified SOD with peroxynitrite-CO2 and the myeloperoxidase system, six newly appearing peptides containing tryptophan derivatives were observed on microLC-ESI-Q-TOF mass analyses and HPLC with a photodiode-array detector. The derivatives of the tryptophan residue exhibiting mass increases of 4, 16 (2 peaks), 32, 45 (major), and 45 Da (minor) were identified as kynurenine, oxindole-3-alanine and its derivatives, dihydroxytryptophan, 6-nitrotryptophan and 5-nitrotryptophan, respectively. We further identified 6-nitrotryptophan from the 1H-NMR spectrum for the pronase-digested product and calculated the yield of 6-nitrotryptophan as being about 30% for each of the modification methods. The tryptophan residue in the modified human Cu,Zn-superoxide dismutase gave the same spectra for the products including 6-nitrotryptophan as the major nitrated product with the two different modification systems.     

Hypoxia followed by reoxygenation induces secretion of cyclophilin A from cultured rat cardiac myocytes

We previously reported that hypoxia followed by reoxygenation (hypoxia/reoxygenation) rapidly activated intracellular signaling such as mitogen-activated protein kinases (MAPKs) including extracellular signal-regulated protein kinase (ERK) 1/2, p38MAPK, and stress-activated protein kinases (SAPKs). To investigate the humoral factors which mediate cardiac response to hypoxia/reoxygenation, we analyzed the conditioned media from cardiac myocytes subjected to hypoxia/reoxygenation by two-dimensional electrophoresis and mass spectrometry. We identified cyclophilin A (CyPA) as one of the proteins secreted from cardiac myocytes in response to hypoxia/reoxygenation. Hypoxia/reoxygenation induced the expression of CyPA and its cell surface receptor CD147 on cardiac myocytes in vitro. This was also confirmed by ischemia/reperfusion in vivo. Recombinant human (rh) CyPA activated ERK1/2, p38MAPK, SAPKs, and Akt in cultured cardiac myocytes. Furthermore, CyPA significantly increased Bcl-2 in cardiac myocytes. These data strongly suggested that CyPA is released from cardiac myocytes in response to hypoxia/reoxygenation and may protect cardiac myocytes from oxidative stress-induced apoptosis.     

The keratan sulfate disaccharide Gal(6S03) beta1,4-GlcNAc(6S03) modulates interleukin 12 production by macrophages in murine Thy-1 type autoimmune disease

It has been reported that disaccharides of the glycosaminoglycans (GAGs), heparin, or heparan sulfate suppress the production of cytokines. Therefore, we examined the effects of GAGs (keratan sulfate, hyaluronan, chondroitin, chondroitin sulfate, and heparin sulfate) disaccharides on production of interleukin (IL)-12, a pivotal cytokine in the Th-1 type immune system. Among the GAG disaccharides, only a keratan sulfate disaccharide, Gal(6-SO(3))-GlcNAc(6-SO(3)) (L4), suppressed IL-12 production in macrophages stimulated with lipopolysaccharides and interferon-gamma. Neither keratan sulfate chains nor keratan sulfate tetrasaccharides elicited any change in the IL-12 production. N-Acetyl-lactosamine, Gal-GlcNAc (LacNAc), also did not change IL-12 production. These results indicated that a certain size, i.e. disaccharide and sulfate, are essential to suppress IL-12 production. L4 was then applied to MRL-lpr/lpr mice, a Th-1 type autoimmune disease model. The treatment of MRL-lpr/lpr mice with L4 1) decreased in serum IL-12, 2) induced apoptosis in T cells in lymph nodes thereby suppressing lymphoaccumulation, and 3) suppressed hypergammaglobulinemia and glomerulonephritis. We showed previously that IL-12 suppresses cell death of T cells, thereby enhancing the lymphoaccumulation in MRL-lpr/lpr mice. Moreover, it has been reported that IL-12 deficiency in MRL-lpr/lpr mice diminishes lymphoaccumulation and delays glomerulonephritis. The treatment with L4 suppressed phosphoprotein kinase C and phosphoinositide 3-kinase expression in macrophages, suggesting that L4 suppresses IL-12 production by inhibiting phosphoprotein kinase C and phosphoinositide 3-kinase pathways.     

Kinetics of ion exchange process for separation of glutamic acid

Kinetics of the separation of L-glutamic acid (GLU) by ion exchange has been studied with strongly acidic H⁺-type cation exchange resin Amberlite IR-122. Since glutamic acid is a trivalent ampholyte and dissociates according to three equilibrium reactions, separation of G⁺ ions by a cation exchange process is accompanied by the dominant reversible reaction, i.e. G⁺+H⁺ ? G⁰. Accompanying reversible reaction has an effect on the ion exchange rate, and decreases the performance of the process comparing with the ideal case that the exchanging ions retain their identity. The analysis was performed first with the ion exchange column, DIC (L/D=0.52); and then with the ion exchange column, IC (L/D=10.9). The data were collected with model glutamic acid solutions for both DIC and IC columns/reactors. IC experimental results were then compared with that of DIC and the effect of scale up on ion exchange process was investigated. The experimental results have provided an adequate basis for the design calculations, and the design parameters were determined. Rate coefficients for the liquid phase mass transfer controlled cation exchange process were calculated and interrelated with a plot of j _Mfactor versus Reynolds number.