Changes in the size of the myocardial damaged area in postischemic reperfusion

The changes in the size of the myocardial injury area during reperfusion after the coronary occlusion-induced ischemia lasting 30 minutes are phasic in nature. Until 3.5 h the injured area increases and after 23.5 h relatively diminishes. After a more prolonged ischemia such manifestations are either unmarked or absent. Ischemia lasting from 30 min to 4 hours followed by reperfusion, as compared with ischemia of the same duration without reperfusion, normally gives rise to the formation of an area of injury, which is less or occasionally equal in size. The data obtained and reported indicate that in the area of coronary occlusion there are groups of cardiomyocytes that differ as regards the resistance to ischemia.     

Induction of endothelial apoptosis by 4-hydroxyhexenal.

Lipid peroxidation and its products such as 4-hydroxy-2-nonenal (HNE) and 4-hydroxyhexenal (HHE) are known to affect redox balance during aging and various degenerative processes, including vascular dysfunction. Deterioration of the endothelial cells that line the vascular wall is known to be an underlying cause of vascular dysfunction. At present, little is known about the mechanism by which HHE induces endothelial cell death (i.e. apoptosis), although HNE-induced apoptotic cell death has been reported. The aim of this study was to determine whether apoptosis induced by HHE in endothelial cells involves peroxynitrite (ONOO(-)). Our results show that in endothelial cells HHE triggers apoptotic cell death by inducing apoptotic Bax coupled with a decrease in anti-apoptotic Bcl-2. Results show that HHE induces reactive oxygen species (ROS), nitric oxide, and ONOO(-) generation, leading to redox imbalance. Furthermore, the antioxidant N-acetyl cysteine, ROS scavenger, and penicillamine, an ONOO(-) scavenger, were found to block HHE-mediated apoptosis. We used confocal laser microscopy to estimate the ability of these inhibitors to attenuate HHE-induced intracellular ONOO(-) levels thus confirming the oxidative mediation of apoptosis in endothelial cells. These findings strongly suggest that accumulated HHE triggers reactive species-mediated endothelial apoptosis, leading to vascular dysfunction as well as vascular aging. During aging, increased lipid peroxidation and its associated production of HHE may exacerbate the weakened redox balance, leading to various chronic degenerative processes including vascular dysfunction.     

Chemotherapy and zoledronate sensitize solid tumour cells to Vγ9Vδ2 T cell cytotoxicity

Combinations of cellular immune-based therapies with chemotherapy and other antitumour agents may be of significant clinical benefit in the treatment of many forms of cancer. Gamma delta (γδ) T cells are of particular interest for use in such combined therapies due to their potent antitumour cytotoxicity and relative ease of generation in vitro. Here, we demonstrate high levels of cytotoxicity against solid tumour-derived cell lines with combination treatment utilizing Vγ9Vδ2 T cells, chemotherapeutic agents and the bisphosphonate, zoledronate. Pre-treatment with low concentrations of chemotherapeutic agents or zoledronate sensitized tumour cells to rapid killing by Vγ9Vδ2 T cells with levels of cytotoxicity approaching 90%. In addition, zoledronate enhanced the chemotherapy-induced sensitization of tumour cells to Vγ9Vδ2 T cell cytotoxicity resulting in almost 100% lysis of tumour targets in some cases. Vγ9Vδ2 T cell cytotoxicity was mediated by perforin following TCR-dependent and isoprenoid-mediated recognition of tumour cells. Production of IFN-γ by Vγ9Vδ2 T cells was also induced after exposure to sensitized targets. We conclude that administration of Vγ9Vδ2 T cells at suitable intervals after chemotherapy and zoledronate may substantially increase antitumour activities in a range of malignancies. Financial support and conflicts of interest: This study was supported by grants from Medinet (Japan), and Suncorp Metway and Gallipoli Research Foundation (Australia). No financial or commercial interests arise from this study. Informed consent: This study was approved by Human Research Ethics Committees of the University of Queensland and Greenslopes Private Hospital and informed consent was obtained from all subjects.     

Selection of mRNA 5'-untranslated region sequence with high translation efficiency through ribosome display

The 5′-untranslated region (5′-UTR) of mRNAs functions as a translation enhancer, promoting translation efficiency. Many in vitro translation systems exhibit a reduced efficiency in protein translation due to decreased translation initiation. The use of a 5′-UTR sequence with high translation efficiency greatly enhances protein production in these systems. In this study, we have developed an in vitro selection system that favors 5′-UTRs with high translation efficiency using a ribosome display technique. A 5′-UTR random library, comprised of 5′-UTRs tagged with a His-tag and Renilla luciferase (R-luc) fusion, were in vitro translated in rabbit reticulocytes. By limiting the translation period, only mRNAs with high translation efficiency were translated. During translation, mRNA, ribosome and translated R-luc with His-tag formed ternary complexes. They were collected with translated His-tag using Ni-particles. Extracted mRNA from ternary complex was amplified using RT-PCR and sequenced. Finally, 5′-UTR with high translation efficiency was obtained from random 5′-UTR library.     

Traits of transgenic Atropa belladonna doubly transformed with different Agrobacterium rhizogenes strains

Hairy root cultures of Atropa belladonna L. were established by infection either with Agrobacterium rhizogenes ATCC 15834 or MAFF 03-01724, and transgenic plants were obtained from both hairy root cultures. Doubly transformed roots were induced by re-infection of the leaf segments of transgenic Atropa belladonna plants (A. rhizogenes 15834) with MAFF 03-01724. Shoots and viviparous leaves were regenerated from the doubly transformed roots. The genetic transformation was determined by the opine assay (agropine, mannopine and/or mikimopine) and polymerase chain reaction. Physiological changes and tropane alkaloid biosynthesis in the hairy roots (singly and doubly transformed) were investigated. The alkaloid content in the doubly transformed root strain was intermediate as compared to the root strains which were singly transformed. On the other hand endogenous IAA levels in doubly transformed roots were significantly decreased compared to both singly transformed roots.Abbreviations BA benzyladenine - IAA indoleacetic acid - NAA naphthaleneacetic acid - PCR polymerase chain reaction - t-ZR trans-zeatin     

Role of charged and hydrophobic residues in the oligomerization of the PYRIN domain of ASC

Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is an adaptor protein composed of two homophilic protein-protein interaction domains, a PYRIN domain (PYD) and a caspase recruitment domain. PYD-dependent oligomerization of ASC is thought to play a crucial role in formation of a molecular platform, the inflammasome, which activates caspase-1. When expressed in cells, the PYD of ASC was shown to form cytoplasmic filaments through self-association. Over 70 single point mutants were analyzed for filament formation in cells expressing the mutant proteins. The set of mutations comprised every single amino acid residue with a charged side chain (Arg, Lys, Asp, and Glu) and a large hydrophobic side chain (Ile, Leu, Met, Phe, Pro, and Val). Filament formation of the ASC PYD was prevented by mutation of Lys21, Leu25, Lys26, Pro40, Arg41, Asp48, and Asp51 of helices 2, 3, and 4. These data identify a coherent interaction surface, establishing a molecular model of PYD-PYD complexes with an important role for charge-charge interactions.     

Electrochemical evaluation of cellular physiological status under stress in Escherichia coli with the rpoS-lacZ reporter gene

We developed an electrochemical detection method for evaluating cellular physiological status based on the stringent response as a means to monitor cell viability. A reporter plasmid was constructed by inserting the beta-galactosidase gene (lacZ) under the control of the rpoS promoter, and then used to transform E. coli cells. Electrochemical responses from the products catalyzed by beta-galactosidase expressed by these E. coli cells were detected using the chronoamperometric technique in a nondestructive manner. Comparisons of response currents between the relA-positive strain and relA-negative strain revealed that increases in these currents were caused by the stringent response due to the stressful alcoholic environment, and thus as a model of stressful cultivating conditions. The current was proportional to the beta-galactosidase activity assayed by a conventional method that required the destruction of cells. The cellular physiological status, which depends on the stringent response as a viability marker, therefore, could then be evaluated online with a current using the rpoS-lacZ reporter gene in the relA-positive strain without pretreatment.     

A Close Relationship between Increases in Galactosyltransferase Activity and the Accumulation of Galactolipids during Plastid Development in Cucumber Seedlings

Changes in the activity of UDP-galactose:diacylglycerol galactosyltransferase(UDGT), a key enzyme in galactolipid biosynthesis, during germinationwere investigated in cucumber (Cucumis sativus L. cv. Aonagajibai)seedlings. After germination, UDGT activity increased duringgrowth in darkness for 4 days, reaching 10 times the activityin ungerminated seeds. Illumination of 4-day-old dark-grownseedlings strongly stimulated the activity. By contrast, inseedlings grown continuously in darkness, the increase in UDGTactivity ceased after 4 days and the activity remained constantthereafter. A similar increase in the specific activity of UDGTwas observed i n the envelope fraction from seedlings, indicatingthat the increase in the enzymatic activity preceded synthesisof other proteins in the envelope membrane. Coincident withthe change in the enzymatic activity, here was an increase inlevels of monogalactosyl diacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), two major constituents of chloroplastmembrane lipids, in the germinated seedlings. Cycloheximideinhibited the light-mediated increase in the enzymatic activityby illumination of 4-day-old dark-grown seedlings, and, as aconsequence, it inhibited the accumulation of MGDG and DGDG.It was clear, therefore, that protein synthesis was necessaryduring this activation. Addition of a cytokinin, benzyladenine(BA), stimulated the increase in the UDGT activity. The increasein the UDGT activity caused by BA was accompanied by the accumulationof galactolipids, as in the case of the activation by light.These results suggest that activation of the final reactionin the synthesis of MGDG, which is catalyzed by the galactosyl-transferase,contributes to the accumulation of galactolipids during thedevelopment of the chloroplast membrane. (Received December 3, 1994; Accepted July 3, 1995)     

Purification and properties of a low-molecular-weight, high-alkaline pectate lyase from an alkaliphilic strain of Bacillus

A low-molecular-weight, high-alkaline pectate lyase (pectate transeliminase, EC 4.2.2.2) was found in an alkaline culture of Bacillus sp. strain KSM-P15, purified to homogeneity, and crystallized. The enzyme had a relative molecular weight of approximately 20,300 as measured by sedimentation equilibrium, with a sedimentation coefficient (s20,w0) of 1.73 S. It was a basic protein with an isoelectric point of pH 10.3, and the alpha-helical content was only 6.6%. In the presence of Ca2+ ions, the enzyme degraded polygalacturonic acid in a random manner to yield 4,5-unsaturated oligo-galacturonides and had its optimal activity around pH 10.5 and 50-55 degrees C. It also had a protopectinase-like activity on cotton fibers. The N-terminal amino acid sequences of the intact protein (28 amino acids) and its two lysyl endopeptidase-cleaved peptide fragments (8 and 12 amino acids) had very low sequence similarity with pectate lyases reported to date. These results strongly suggest that the pectate lyase of Bacillus sp. strain KSM-P15 may be a novel enzyme and belongs in a new family.