A novel role for ursodeoxycholic acid in inhibiting apoptosis by modulating mitochondrial membrane perturbation

The hydrophilic bile salt ursodeoxycholic acid (UDCA) protects against the membrane-damaging effects associated with hydrophobic bile acids. This study was undertaken to (a) determine if UDCA inhibits apoptosis from deoxycholic acid (DCA), as well as from ethanol, TGF-beta1, Fas ligand, and okadaic acid; and to (b) determine whether mitochondrial membrane perturbation is modulated by UDCA. DCA induced significant hepatocyte apoptosis in vivo and in isolated hepatocytes determined by terminal transferase-mediated dUTP-digoxigenin nick end-labeling assay and nuclear staining, respectively (P < 0.001). Apoptosis in isolated rat hepatocytes increased 12-fold after incubation with 0.5% ethanol (P < 0.001). HuH-7 cells exhibited increased apoptosis with 1 nM TGF-beta1 (P < 0. 001) or DCA at >/= 100 microM (P < 0.001), as did Hep G2 cells after incubation with anti-Fas antibody (P < 0.001). Finally, incubation with okadaic acid induced significant apoptosis in HuH-7, Saos-2, Cos-7, and HeLa cells. Coadministration of UDCA with each of the apoptosis-inducing agents was associated with a 50-100% inhibition of apoptotic changes (P < 0.001) in all the cell types. Also, UDCA reduced the mitochondrial membrane permeability transition (MPT) in isolated mitochondria associated with both DCA and phenylarsine oxide by > 40 and 50%, respectively (P < 0.001). FACS(R) analysis revealed that the apoptosis-inducing agents decreased the mitochondrial transmembrane potential and increased reactive oxygen species production (P < 0.05). Coadministration of UDCA was associated with significant prevention of mitochondrial membrane alterations in all cell types. The results suggest that UDCA plays a central role in modulating the apoptotic threshold in both hepatocytes and nonliver cells, and inhibition of MPT is at least one pathway by which UDCA protects against apoptosis.     

A study of the lethal and sublethal toxicity of polyphase P-100, an antisapstain fungicide containing 3-iodo-2-propynyl butyl carbamate (IPBC), on fish and aquatic invertebrates

Since pneumotachograph masks are commonly used in studies of speech breathing, the purpose of this study was to measure the differences in respiratory volumetric and frequency measures during speech under two conditions: with and without a circumferentially vented pneumotachograph mask coupled to the face. Thus we sought to identify whether changes in breathing patterns occur with the use of a specific face mask, because these patterns are accepted as representative of normal speech breathing. Subjects were 10 normal-speaking women, each of whom produced a syllable train and a connected speech task, both at comfortable intensity levels. Respiratory measures were made using linearized magnetometers during speech production. The measurements included lung volume, rib cage volume, and abdominal volume at utterance initiation and termination, volume excursions during the utterance, and the number of breath groups during the speech task. There were no significant differences between the mask-on and mask-off conditions in volumetric and frequency measures. A significant task difference for abdominal initiation was found. It was concluded that the use of a circumferentially vented pneumotachograph mask does not alter the reliability of respiratory volume and frequency measures for studies of voice.     

Redefining reductive sulfate assimilation in higher plants: a role for APS reductase, a new member of the thioredoxin superfamily?

The reaction steps leading from the intermediate adenosine 5'-phosphosulfate (APS) to sulfide within the higher plant reductive sulfate assimilation pathway are the subject of controversy. Two pathways have been proposed: a 'bound intermediate' pathway in which the sulfo group of APS is first transferred by APS sulfotransferase to a carrier molecule to form a bound sulfite intermediate and is then further reduced by thiosulfonate reductase to bound sulfide; and a 'free intermediate' pathway in which APS is further activated to 3'-phosphoadenosine 5'-phosphosulfate (PAPS) by APS kinase followed by reduction of the sulfo group to free sulfite by PAPS reductase. Sulfite is then reduced to free sulfide by sulfite reductase. Sulfide, either free or bound, is then incorporated into organic form (as cysteine) by the enzyme O-acetylserine (thiol) lyase. In order to better characterize the pathway we attempted to clone PAPS reductase cDNAs by functional complementation of an Escherichia coli cysH mutant to prototrophy. We found no evidence for PAPS reductase cDNAs but did identify cDNAs that encode a small family of novel, chloroplast-localized proteins with APS reductase activity that are new members of the thioredoxin superfamily. We show here that the thioredoxin domain of these proteins is functional. We speculate that rather than proceeding via either of the pathways proposed above, reductive sulfate assimilation proceeds via the reduction of APS to sulfite by APS reductase and the subsequent reduction of sulfite to sulfide by sulfite reductase. In this scheme the product of the APS kinase reaction, PAPS, is not a direct intermediate in the pathway but rather acts as a substrate for sulfotransferase action and perhaps as a store of activated sulfate that can be returned to the pathway as APS via phosphohydrolase action on PAPS. Interactions between enzyme isoforms within the chloroplast stroma may bring about substrate channeling of APS and contribute to the partitioning of APS between sulfotransferase reactions on the one hand and the synthesis of cysteine and related metabolites via the reductive sulfate assimilation pathway on the other.     

Heat shock provides delayed protection against oxidative injury in cultured human umbilical vein endothelial cells

During both mild and severe ischemia, vascular endothelial cells lining large and small vessels of the ischemic organ are exposed to oxygen-derived free radicals resulting in oxidative damage to the organ. Heat shock has been shown to induce thermotolerance and also protect against ischemic injury, possibly via increased synthesis of heat shock proteins (HSPs). We hypothesized that heat shock preconditioning may protect human endothelial cells against oxidative damage. Cultured human umbilical vein endothelial cells (HUVEC) were subjected to heat shock (42 degrees C, 1 h) and allowed to recover for 2 or 20 h, at which times the cells were oxidatively stressed for 1 h by exposing them to 100-200 mumol/l of hydrogen peroxide (H2O2). Cellular damage was assessed immediately and 18 h later by morphology and release of lactate dehydrogenase (LDH). No protection of HUVEC was seen using the 2-hour recovery interval, but a significant protection (P < 0.05) was observed after the 20-hour delay. Northern blot analysis at 1 and 2 h after heating showed induction of HSP-70 mRNA. Western blot analysis demonstrated a significant increase in HSP-72 protein after 2 as well as 20 h of recovery from heat shock, although the amounts of protein at the two times were not significantly different. Furthermore, no differences in the activity of the antioxidant enzyme catalase were observed between heated and unheated HUVEC at 2 and 20 h after heat preconditioning. Thus, heat shock preconditioning induces delayed protection against oxidative injury in HUVEC, and the mechanism of protection appears to involve more than the expression of HSP-72 or activity of catalase.     

Localization of a baculovirus-induced chitinase in the insect cell endoplasmic reticulum

Confocal immunofluorescence microscopy was used to demonstrate that the Autographa californica nucleopolyhedrovirus (AcMNPV) chitinase was localized within the endoplasmic reticulum (ER) of virus-infected insect cells. This was consistent with removal of the signal peptide from the chitinase and an ER localization motif (KDEL) at the carboxyl end of the protein. Chitinase release from cells, a prerequisite for liquefaction of virus-infected insect larvae, appears to be aided by synthesis of the p10 protein. Deletion of p10 from the AcMNPV genome delayed the appearance of chitinase activity in the medium of virus-infected cells by 24 h and also delayed liquefaction of virus-infected Trichoplusia ni larvae by the same period.     

Repressor titration: a novel system for selection and stable maintenance of recombinant plasmids

The propagation of recombinant plasmids in bacterial hosts, particularly in Escherichia coli, is essential for the amplification and manipulation of cloned DNA and the production of recombinant proteins. The isolation of bacterial transformants and subsequent stable plasmid maintenance have traditionally been accomplished using plasmid-borne selectable marker genes. Here we describe a novel system that employs plasmid-mediated repressor titration to activate a chromosomal selectable marker, removing the requirement for a plasmid-borne marker gene. A modified E.coli host strain containing a conditionally essential chromosomal gene (kan) under the control of the lac operator/promoter, lac O/P, has been constructed. In the absence of an inducer (allolactose or IPTG) this strain, DH1 lackan , cannot grow on kanamycin-containing media due to the repression of kan expression by LacI protein binding to lac O/P. Transformation with a high copy-number plasmid containing the lac operator, lac O, effectively induces kan expression by titrating LacI from the operator. This strain thus allows the selection of plasmids without antibiotic resistance genes (they need only contain lac O and an origin of replication) which have clear advantages for use as gene therapy vectors. Regulation in the same way of an essential, endogenous bacterial gene will allow the production of recombinant therapeutics devoid of residual antibiotic contamination.     

Insertion element IS3-based PCR method for subtyping Escherichia coli O157:H7

An Escherichia coli O157:H7 subtyping method based on PCR amplification of variable DNA sequences between the repetitive element IS3 was developed. Template DNA was prepared by boiling cells in Chelex. Two separate IS3 PCR amplifications were performed for each isolate: one with a single primer (primer IS3A) and one with two primers (primers IS3A and IS3B). The IS3 PCR subtyping method was applied to 35 epidemiologically related and unrelated E. coli O157:H7 isolates that had been previously characterized by pulsed-field gel electrophoresis (PFGE). PFGE identified 25 different subtypes (difference of one or more bands). PCR with single primer IS3A and primer pair IS3A-IS3B identified 6 and 14 different subtypes, respectively. By combining the results of the two PCR amplifications, 15 different IS3 PCR subtypes were identified. While not as sensitive as PFGE, IS3 PCR subtyping grouped all outbreak-related isolates. IS3 PCR banding patterns were reproducible between amplifications and between subcultures. IS3 PCR could serve as a simple, rapid screening method for the identification of unrelated E. coli O157:H7 isolates.     

Characterization and screening for mutations of the growth arrest-specific 11 (GAS11) and C16orf3 genes at 16q24.3 in breast cancer

BACKGROUND: Both fibroblast-mediated cytokine gene therapy and bone marrow transplantation (BMT) have proven to be efficient protocols for the recovery of bone marrow depression. In this report, the effects of fibroblast-mediated interleukin (IL)-6 gene therapy, in combination with BMT, on the recovery of irradiation-induced bone marrow depression were investigated. METHODS: NIH3T3 fibroblast cells engineered to secrete IL-6 (NIH3T3-IL-6) or NIH3T3 cells transduced with the neomycin gene (NIH3T3-Neo), in combination with 10(7), 10(6), or 10(5) syngeneic bone marrow cells, were implanted into irradiated mice. RESULTS: The platelets and white blood cells in the peripheral blood of the irradiated mice increased greatly 12 days after implantation of NIH3T3-IL-6 cells and BMT, the white blood cell counts were restored to a normal level 32 days after the combined therapy, and the platelet number was obviously higher than that in mice implanted with NIH3T3-Neo and BMT. Twenty and 25 days after the combined therapy, the mice showed accelerated recovery of colony-forming unit (CFU)-granulocyte/macrophages and CFU-megakaryocytes when compared with the mice implanted with NIH3T3-Neo cells and BMT. Ten days after lethal irradiation with gamma rays, the spleens formed more CFU-spleen in mice implanted with NIH3T3-IL-6 cells and BMT than in mice injected with phosphate-buffered saline or NIH3T3-Neo cells. Combined therapy with NIH3T3-IL-6 cell implantation and BMT delayed the survival period of the hematopoietic-depressed mice significantly when compared with therapy with NIH3T3-Neo cell implantation and BMT. CONCLUSIONS: These data demonstrated that the combined therapy of fibroblast-mediated IL-6 gene therapy and BMT could significantly promote the recovery of irradiation-induced hematopoietic depression.     

Deficient insulin-like growth factor I in chronic heart failure predicts altered body composition, anabolic deficiency, cytokine and neurohormonal activation

BACKGROUND: Recent studies of growth hormone supplementation in chronic heart failure have been associated with variable results. Acquired abnormalities of biochemical parameters of the growth hormone insulin-like growth factor I axis have been associated with severe chronic heart failure. There are suggestions of an acquired growth hormone resistance with deficient insulin-like growth factor I in some patients. OBJECTIVES: Therefore, we set out to investigate the clinical and functional status and the degree of cytokine and neurohormonal alteration of chronic heart failure patients with deficient insulin-like growth factor I responses. METHODS: Patients with chronic heart failure were divided into two groups according to their insulin-like growth factor I levels (classified according to the manufacturer's assay range in normal controls): low insulin-like growth factor I <104 (n = 20; 89 +/- 9.6 ng/ml), and normal/high >104 ng/ml (n = 32; 169 +/- 52 ng/ml). Between groups there was no difference in age (low versus high: 65.3 +/- 12.1 versus 61.6 +/- 9.1 years, p = 0.21), body mass index, aerobic capacity (peak oxygen consumption: low versus high: 15.5 +/- 5.2 versus 17.3 +/- 6.3 mL/kg/min, p = 0.23), left ventricular ejection fraction, New York Heart Association classification. RESULTS: During quadriceps strength testing, patients with low insulin-like growth factor I had reduced absolute strength (-24%), and strength per unit area muscle (- 14%) than patients with normal/high insulin-like growth factor I. Leg muscle cross-sectional area was lower in the low insulin-like growth factor I group (-12% and -13% for right and left legs, respectively). These alterations were accompanied by increased levels of growth hormone (+145%), tumor necrosis factor-alpha (+46%), cortisol/ dehydroepiandrosterone ratio (+60%), noradrenaline (+49%) and adrenaline (+136%) (all at least p < 0.05). CONCLUSIONS: Patients with low insulin-like growth factor I levels show signs of altered body composition, cytokine and neuroendocrine activation, to a greater extent than patients with normal/high levels.