Genomic analyses of germline and somatic variation in high-grade serous ovarian cancer.

BACKGROUND: High-grade serous ovarian cancers (HGSCs) display a high degree of complex genetic alterations. In this study, we identified germline and somatic genetic alterations in HGSC and their association with relapse-free and overall survival. Using a targeted capture of 557 genes involved in DNA damage response and PI3K/AKT/mTOR pathways, we conducted next-generation sequencing of DNA from matched blood and tumor tissue from 71 HGSC participants. In addition, we performed the OncoScan assay on tumor DNA from 61 participants to examine somatic copy number alterations (SCNA). RESULTS: Approximately one-third of tumors had loss-of-function (LOF) germline (18/71, 25.4%) or somatic (7/71, 9.9%) variants in the DNA homologous recombination repair pathway genes BRCA1, BRCA2, CHEK2, MRE11A, BLM, and PALB2. LOF germline variants also were identified in other Fanconi anemia genes and in MAPK and PI3K/AKT/mTOR pathway genes. Most tumors harbored somatic TP53 variants (65/71, 91.5%). Using the OncoScan assay on tumor DNA from 61 participants, we identified focal homozygous deletions in BRCA1, BRCA2, MAP2K4, PTEN, RB1, SLX4, STK11, CREBBP, and NF1. In total, 38% (27/71) of HGSC patients harbored pathogenic variants in DNA homologous recombination repair genes. For patients with multiple tissues from the primary debulking or from multiple surgeries, the somatic mutations were maintained with few newly acquired point mutations suggesting that tumor evolution was not through somatic mutations. There was a significant association of LOF variants in homologous recombination repair pathway genes and high-amplitude somatic copy number alterations. Using GISTIC analysis, we identified NOTCH3, ZNF536, and PIK3R2 in these regions that were significantly associated with an increase in cancer recurrence and a reduction in overall survival. CONCLUSIONS: From 71 patients with HGCS, we performed targeted germline and tumor sequencing and provided a comprehensive analysis of these 557 genes. We identified germline and somatic genetic alterations including somatic copy number alterations and analyzed their associations with relapse-free and overall survival. This single-site long-term follow-up study provides additional information on genetic alterations related to occurrence and outcome of HGSC. Our findings suggest that targeted treatments based on both variant and SCNA profile potentially could improve relapse-free and overall survival.

Genomic Analyses of Germline and Somatic Variation in High-Grade Serous Ovarian Cancer.

Background High-grade serous ovarian cancers (HGSCs) display a high degree of complex genetic alterations. In this study, we identified germline and somatic genetic alterations in HGSC and their association with relapse-free and overall survival. Using a targeted capture of 577 genes involved in DNA damage response and PI3K/AKT/mTOR pathways, we conducted next-generation sequencing of DNA from matched blood and tumor tissue from 71 HGSC participants. In addition, we performed the OncoScan assay on tumor DNA from 61 participants to examine somatic copy number alterations. Results Approximately one-third of tumors had loss-of-function germline (18/71, 25.4%) or somatic (7/71, 9.9%) variants in the DNA homologous recombination repair pathway genes BRCA1, BRCA2, CHEK2, MRE11A, BLM , and PALB2 . Loss-of-function germline variants also were identified in other Fanconi anemia genes and in MAPK and PI3K/AKT/mTOR pathway genes. Most tumors harbored somatic TP53 variants (65/71, 91.5%). Using the OncoScan assay on tumor DNA from 61 participants, we identified focal homozygous deletions in BRCA1, BRCA2, MAP2K4, PTEN, RB1, SLX4, STK11, CREBBP , and NF1 . In total, 38% (27/71) of HGSC patients harbored pathogenic variants in DNA homologous recombination repair genes. For patients with multiple tissues from the primary debulking or from multiple surgeries, the somatic mutations were maintained with few newly acquired point mutations suggesting that tumor evolution was not through somatic mutations. There was a significant association of loss-of-function variants in homologous recombination repair pathway genes and high-amplitude somatic copy number alterations. Using GISTIC analysis, we identified NOTCH3, ZNF536 , and PIK3R2 in these regions that were significantly associated with an increase in cancer recurrence and a reduction in overall survival. Conclusions From 71 patients with HGCS, we performed targeted germline and tumor sequencing and provided a comprehensive analysis of these 577 genes. We identified germline and somatic genetic alterations including somatic copy number alterations and analyzed their associations with relapse-free and overall survival. This single-site long-term follow-up study provides additional information on genetic alterations related to occurrence and outcome of HGSC. Our findings suggest that targeted treatments based on both variant and SCNA profile potentially could improve relapse-free and overall survival.

Effectiveness of Perioperative Immunologic Markers Monitoring for Predicting Early Acute Cellular Rejection After Living Donor Liver Transplantation.

BACKGROUND: The objective of this study was to determine whether perioperative immunologic markers monitoring could predict early acute cellular rejection (ACR) after living donor liver transplantation (LDLT). MATERIALS AND METHODS: From September 2010 to June 2013, a total of 172 patients underwent LDLT at our transplant center. Of them, 26 patients were excluded because of infection. We retrospectively reviewed the remaining 146 patients. CD4 lymphocyte activity, T cell subsets test, and serum cytokine panel were checked on the day before transplantation and at 20 days after transplantation. These patients were divided into 3 groups: 1. normal liver function test (LFT) group; 2. increased LFT without rejection group; and 3. early ACR group. We excluded the increased LFT without rejection group in order to rule out multiple factors influencing immunologic factors. RESULTS: CD4 lymphocyte activity (P = .004) was significantly increased while CD4+/CD25+/FOXP3+ cells (P < .001) and interleukin (IL)-17 (P = .002) levels were significantly decreased during the perioperative period. Pretransplant IL-6 (P = .014) and IL-17 (P = .029) levels in the early ACR group were significantly lower than those in the normal LFT group. The proportion of patients with increased IL-6 during perioperative period in the early ACR group was higher than that in the normal LFT group, although the difference was not statistically significant (P = .065). CONCLUSION: Our results suggest that IL-6 and IL-17 levels are associated with early ACR in LDLT patients. However, whether monitoring perioperative immunologic markers could predict early ACR remains unclear. Further prospective studies are needed to reach a definite conclusion.

Modulation of 1-methyl-4-phenylpyridinium-induced mitochondrial dysfunction and cell death in PC12 cells by K(ATP) channel block.

The present study investigated the effect of 5-hydroxydecanoate, a selective mitochondrial K(ATP) channel blocker, on the cytotoxicity of neurotoxin 1-methyl-4-phenylpyridinium (MPP(+)) in differentiated PC12 cells. 5-Hydroxydecanoate and glibenclamide (a cell surface and mitochondrial K(ATP) channel inhibitor) reduced the MPP(+)-induced cell death and GSH depletion and showed a maximal inhibitory effect at 5 and 10 microM, respectively. Addition of 5-hydroxydecanoate attenuated the MPP(+)-induced nuclear damage, changes in the mitochondrial membrane permeability and increase in the reactive oxygen species formation in PC12 cells. The results show that 5-hydroxydecanote may prevent the MPP(+)-induced viability loss in PC12 cells by suppressing formation of the mitochondrial permeability transition, leading to the cytochrome c release and caspase-3 activation. This effect appears to be accomplished by the inhibitory action on the formation of reactive oxygen species and the depletion of GSH. The blockade of mitochondrial K(ATP) channels seems to prevent the MPP(+)-induced neuronal cell damage.

Characteristics and smoking cessation outcomes of patients returning for repeat tobacco dependence treatment.

Previous studies of tobacco dependence treatment have reported very low cessation rates among smokers who relapse and return to make a subsequent formal attempt to quit. This retrospective cohort study examined 1745 patients who attended a tobacco dependence clinic between 2001 and 2005, and the characteristics and outcomes of those who relapsed and returned for repeat treatment. Patients who returned for repeat treatment showed higher markers of nicotine dependence and were more likely to have a history of treatment for mental health problems than patients who attended the clinic for only one treatment episode. Among patients who relapsed and returned for repeat treatment, the 26-week abstinence rates were similar for each consecutive quit attempt (23%, 22% and 20%). Clinicians should encourage smokers who relapse after an initial treatment episode to return for treatment, and repeat treatment should focus on addressing high nicotine dependence and potentially co-occurring mental health problems in order to improve cessation outcomes.

7-Ketocholesterol enhances 1-methyl-4-phenylpyridinium-induced mitochondrial dysfunction and cell death in PC12 cells.

The present study investigated the promoting effect of oxysterol 7-ketocholesterol against the cytotoxicity of 1-methyl-4-phenylpyridinium (MPP(+)) in differentiated PC12 cells. 7-Ketocholesterol significantly enhanced the MPP(+)-induced nuclear damage, decrease in the mitochondrial transmembrane potential, cytosolic accumulation of cytochrome c, activation of caspase-3, increase in the formation of reactive oxygen species and depletion of GSH. N-Acetylcysteine, ascorbate, trolox, carboxy-PTIO and Mn-TBAP reduced the cytotoxic effect of MPP(+) in the presence of 7-ketocholesterol. The results indicate that 7-ketocholesterol shows a synergistic effect against the cytotoxic effect of MPP(+). 7-Ketocholesterol may enhance the MPP(+)-induced viability loss in PC12 cells by promoting the mitochondrial membrane permeability change, release of cytochrome c and subsequent activation of caspase-3, which is associated with the increased formation of reactive oxygen species and depletion of GSH. The findings suggest that 7-ketocholesterol as a promoting agent for the formation of mitochondrial permeability transition may enhance the toxic neuronal cell injury.

Food restriction enhances endogenous and corticotropin-induced plasma elevations of free but not total corticosterone throughout life in rats.

Chronic food restriction (FR), which retards many aging processes, enhances the endogenous diurnal peak of plasma total corticosterone (B) in young rats. Although the FR-dependent enhancement of total B disappears in aged rats, increased levels of the bioavailable fraction, free B, appear to be maintained. In young rats, we previously found that the FR-induced increase in the diurnal peak of total B is associated with increased adrenal response to corticotropin, also known as adrenocorticotropic hormone (ACTH). Here we show that the FR-enhanced adrenal response of total B to ACTH disappears with age but that the enhanced response of free B is maintained. We measured the endogenous diurnal peak and the response to ACTH of total and free B in 10-, 16-, and 22-month-old ad-libitum fed and FR male Fischer 344 rats in the afternoon, when plasma B peaks. At 10 and 16 months, FR rats showed enhanced total plasma B responses to ACTH relative to ad-libitum fed rats, but not at 22 months. By contrast, the response of free B to ACTH was enhanced by FR at all ages. The effect of FR on patterns of endogenous total and free diurnal B in these three age groups paralleled the ACTH-response data. The enhanced adrenocortical response of FR rats to ACTH does not reflect an increased expression of ACTH-receptor (ACTH-R) mRNA, because ACTH-R mRNA/microg adrenal RNA and ACTH-R mRNA/mg adrenal weight did not differ between ad-libitum fed and FR rats at any age.

Protective effect of harmaline and harmalol against dopamine- and 6-hydroxydopamine-induced oxidative damage of brain mitochondria and synaptosomes, and viability loss of PC12 cells.

The present study elucidated the protective effect of beta-carbolines (harmaline, harmalol and harmine) against oxidative damage of brain mitochondria, synaptosomes and PC12 cells induced by either dopamine or 6-hydroxydopamine. Harmaline, harmalol and antioxidant enzymes (superoxide dismutase/SOD and catalase) decreased the alteration of mitochondrial swelling and membrane potential induced by 200 microM dopamine or 100 microM 6-hydroxydopamine. Deprenyl attenuated the dopamine-induced mitochondrial dysfunction but did not reduce the effect of 6-hydroxydopamine. While beta-carbolines inhibited the electron flow in mitochondria, they did not enhance the depressant effect of catecholamines. beta-Carbolines and antioxidant enzymes reversed the depression of synaptosomal Ca2+ uptake induced by 10 microM catecholamines. The compounds inhibited the catecholamine-induced thioredoxin reductase inhibition, thiol oxidation and carbonyl formation in mitochondria and synaptosomes. beta-Carbolines decreased the reactive species-induced deoxyribose degradation. Harmaline and harmalol reduced the catecholamine-induced loss of the transmembrane potential and of cell viability in PC12 cells. beta-Carbolines alone did not show a significant cytotoxic effect on PC12 cells. The results suggest that beta-carbolines may attenuate the dopamine- or 6-hydroxydopamine-induced alteration of brain mitochondrial and synaptosomal functions, and viability loss in PC12 cells, by a scavenging action on reactive oxygen species and inhibition of thiol oxidation.

Food restriction differentially affects pituitary hormone mRNAs throughout the adult life span of male F344 rats.

Because neuroendocrine mechanisms may contribute to the antiaging effects of food restriction (FR), we measured the effect of FR on mRNAs encoding anterior pituitary (AP) tropic hormones. Slot blots or RNase protection assays were done on AP RNA from 3-, 6-, 12-, 18- and 24-mo-old male F344 rats consuming food ad libitum (AL) or food restricted (FR; to 60% of AL food intake) from 6 wk. Both AL and FR rats gained body weight during the study (P < 0.05), but FR rats weighed approximately 40% less (P < 0.0001). Messenger RNA levels were expressed in two ways, i.e., per total AP and per microgram total AP RNA. Proopiomelanocortin (POMC) mRNA/microg RNA was higher (P < 0.0005) in FR than in AL rats at all ages. Thyroid-stimulating hormone (TSH) beta mRNA declined with age (P < 0.05) in AL but not FR rats and was reduced by FR up to 12 mo (P < 0.01). Growth hormone (GH) mRNA/microg RNA declined with age (P < 0.05) in AL but not FR rats, and total GH mRNA in the AP was reduced by FR at early ages (P < 0.05). FR reduced prolactin (PRL) mRNA and its age-related increase (P < 0.0005). Levels of luteinizing hormone (LH) beta and follicle-stimulating hormone (FSH) beta mRNAs did not differ between AL and FR rats until 12 mo, but thereafter rose in FR (LH beta mRNA; P < 0.01, FSH beta mRNA; P < 0.05). Many of these changes in gene expression corroborate previously reported hormonal changes in FR rodents and mutant mice with extended life spans, and thus provide further support for the hypothesis that an altered hormonal milieu contributes to the antiaging effects of food restriction.

Effect of iron and ascorbate on cyclosporine-induced oxidative damage of kidney mitochondria and microsomes.

The stimulatory effect of iron and ascorbate on the damaging action of cyclosporine in kidney mitochondria, microsomes and epithelial cells was examined. Cyclosporine induced malondialdehyde formation and hydrogen peroxide production in mitochondria and attenuated the activity of MnSOD and glutathione peroxidase. The damaging effect of cyclosporine (50 microM) plus Fe2+(20 microM) on mitochondrial and microsomal lipids and proteins as well as mitochondrial thiols was greater than the summation of the oxidizing action of cyclosporine alone and Fe2+ alone. As for tissue components, iron enhanced cyclosporine-induced viability loss in kidney epithelial cells. Fe2+, EDTA and H2O2- induced 2-alpha deoxyribose degradation was attenuated by 10 mM DMSO and 200 microM DTPA but not affected by 200 microM cyclosporine. The addition of Fe2+ caused a change in the absorbance spectrum of cyclosporine in the wavelength range 230-350 nm. The simultaneous addition of cyclosporine (50 microM) and ascorbate (100 microM) showed the enhanced peroxidative effect on mitochondrial and microsomal lipids, which was inhibited by DTPA and EDTA (1 mM). Similar to iron, ascorbate enhanced cyclosporine-induced cell viability loss. The results show that iron and ascorbate promote the damaging action of cyclosporine in kidney cortex mitochondria and microsomes and in kidney epithelial cells, which may contribute to the enhancement of cyclosporine-induced nephrotoxicity.

Differential regulation of protein tyrosine kinase on free radical production, granule enzyme release, and cytokine synthesis by activated murine peritoneal macrophages.

The present study examined the regulatory effect of tyrosine kinase inhibitors (genistein, tyrphostin, and 2,5-dihydroxycinnamate) on the free radical production, granule enzyme release, and synthesis of interleukin (IL)-8 and granulocyte macrophage-colony stimulating factor (GM-CSF) in murine peritoneal macrophages exposed to different stimulators [10 ng/mL of IL-1, 1 microgram/mL of lipopolysaccharide (LPS), and 1 microM N-formyl-methionyl-leucyl-phenylalanine (fMLP)]. Protein tyrosine kinase (PTK) inhibitors attenuated the stimulated superoxide, hydrogen peroxide, and nitric oxide production in macrophages stimulated with IL-1, LPS, or fMLP. N,N-Dimethylsphingosine (DMS) alone stimulated superoxide and hydrogen peroxide production by intact macrophages, but at 45 microM the stimulatory effect on superoxide production was not found. In contrast, DMS attenuated nitric oxide production by macrophages. High concentrations of DMS, tyrphostin, and 2,5-dihydroxycinnamate showed cytotoxic effects. PTK inhibitors did not exhibit a significant effect on granule enzyme release induced by IL-1, whereas they attenuated the effect of LPS and fMLP on degranulation. Genistein and tyrphostin decreased the production of IL-8 and GM-CSF in macrophages activated by IL-1, whereas 2,5-dihydroxycinnamate did not affect it. The results suggest that tyrosine kinases exposed to IL-1, LPS, and fMLP may exert different modulatory actions on macrophage responses. The IL-1-activated macrophage responses, particularly degranulation, appear to be differently regulated by tyrosine kinases compared with the responses activated by LPS and fMLP.

Protective effect of boldine on oxidative mitochondrial damage in streptozotocin-induced diabetic rats.

Increased oxidative stress has been suggested to be involved in the pathogenesis and progression of diabetic tissue damage. Several antioxidants have been described as beneficial for oxidative stress-associated diseases. Boldine ([s]-2,9-dihydroxy-1, 10-dimethoxyaporphine) is a major alkaloid found in the leaves and bark of boldo (Peumus boldus Molina), and has been shown to possess antioxidant activity and anti-inflammatory effects. From this point of view, the possible anti-diabetic effect of boldine and its mechanism were evaluated. The experiments were performed on male rats divided into four groups: control, boldine (100 mg kg(-1), daily in drinking water), diabetic [single dose of 80 mg kg(-1)of streptozotocin (STZ), i.p.] and diabetic simultaneously fed with boldine for 8 weeks. Diabetic status was evaluated periodically with changes of plasma glucose levels and body weight in rats. The effect of boldine on the STZ-induced diabetic rats was examined with the formation of malondialdehydes and carbonyls and the activities of endogenous antioxidant enzymes (superoxide dismutase and glutathione peroxidase) in mitochondria of the pancreas, kidney and liver. The scavenging action of boldine on oxygen free radicals and the effect on mitochondrial free-radical production were also investigated. The treatment of boldine attenuated the development of hyperglycemia and weight loss induced by STZ injection in rats. The levels of malondialdehyde (MDA) and carbonyls in liver, kidney and pancreas mitochondria were significantly increased in STZ-treated rats and decreased after boldine administration. The activities of mitochondrial manganese superoxide dismutase (MnSOD) in the liver, pancreas and kidney were significantly elevated in STZ-treated rats. Boldine administration decreased STZ-induced elevation of MnSOD activity in kidney and pancreas mitochondria, but not in liver mitochondria. In the STZ-treated group, glutathione peroxidase activities decreased in liver mitochondria, and were elevated in pancreas and kidney mitochondria. The boldine treatment restored the altered enzyme activities in the liver and pancreas, but not the kidney. Boldine attenuated both STZ- and iron plus ascorbate-induced MDA and carbonyl formation and thiol oxidation in the pancreas homogenates. Boldine decomposed superoxide anions, hydrogen peroxides and hydroxyl radicals in a dose-dependent manner. The alkaloid significantly attenuated the production of superoxide anions, hydrogen peroxide and nitric oxide caused by liver mitochondria. The results indicate that boldine may exert an inhibitory effect on STZ-induced oxidative tissue damage and altered antioxidant enzyme activity by the decomposition of reactive oxygen species and inhibition of nitric oxide production and by the reduction of the peroxidation-induced product formation. Boldine may attenuate the development of STZ-induced diabetes in rats and interfere with the role of oxidative stress, one of the pathogeneses of diabetes mellitus.

Protective effect of harmalol and harmaline on MPTP neurotoxicity in the mouse and dopamine-induced damage of brain mitochondria and PC12 cells.

The present study elucidated the protective effect of beta-carbolines (harmaline, harmalol, and harmine) on oxidative neuronal damage. MPTP treatment increased activities of total superoxide dismutase, catalase, and glutathione peroxidase and levels of malondialdehyde and carbonyls in the basal ganglia, diencephalon plus midbrain of brain compared with control mouse brain. Coadministration of harmalol (48 mg/kg) attenuated the MPTP effect on the enzyme activities and formation of tissue peroxidation products. Harmaline, harmalol, and harmine attenuated both the 500 microM MPP(+)-induced inhibition of electron flow and membrane potential formation and the 100 microM dopamine-induced thiol oxidation and carbonyl formation in mitochondria. The scavenging action of beta-carbolines on hydroxyl radicals was represented by inhibition of 2-deoxy-D-ribose degradation. Harmaline and harmalol (100 microM) attenuated 200 microM dopamine-induced viability loss in PC12 cells. The beta-carbolines (50 microM) attenuated 50 microM dopamine-induced apoptosis in PC12 cells. The compounds alone did not exhibit significant cytotoxic effects. The results indicate that beta-carbolines attenuate brain damage in mice treated with MPTP and MPP(+)-induced mitochondrial damage. The compounds may prevent dopamine-induced mitochondrial damage and PC12 cell death through a scavenging action on reactive oxygen species and inhibition of monoamine oxidase and thiol oxidation.

Depressant effects of ambroxol on lipopolysaccharide- or fMLP-stimulated free radical production and granule enzyme release by alveolar macrophages.

In order to explore the depressant action of ambroxol, a bronchial expectorant, on the activated alveolar macrophage responses, its effect on lipopolysaccharide (LPS)- or N-formyl-methionyl-leucyl-phenylalanine (fMLP)- stimulated free radical production and granule enzyme release by rat lung alveolar macrophages was investigated. Ambroxol attenuated the 100 ng/ml LPS- or 1 microM fMLP-stimulated superoxide, H(2)O(2)and nitric oxide production and releases of acid phosphatase and lysozyme by alveolar macrophages. Ambroxol attenuated phorbol myristate acetate-stimulated superoxide and nitric oxide production that was inhibited by 100 nM staurosporine. N,N-dimethylsphingosine (DMS, 4.5 and 9 microM) alone stimulated superoxide production by macrophages, while 45 microM of the compound did not show a stimulatory effect. However, DMS decreased nitric oxide production in a dose-dependent manner. Ambroxol did not alter the DMS effect on free radical production that was affected by 10 microM genistein. A preincubation of macrophages with ambroxol (10 and 100 microM), staurosporine and genistein attenuated the elevation of [Ca(2+)](i)caused by LPS. The results suggest that ambroxol exerts a depressant effect on LPS- or fMLP-stimulated free radical production and granule enzyme release by rat alveolar macrophages, which may be attributed to its inhibitory action on the activation process, protein kinase C, but its action on protein tyrosine kinase is not suggested.

The inhibitory effect of ambroxol on hypochlorous acid-induced tissue damage and respiratory burst of phagocytic cells.

Ambroxol (100 microM and 1 mM) and the thiols (all 1 mM), glutathione, tiopronin and cysteine, significantly attenuated the myeloperoxidase, H(2)O(2) and Cl(-) system-caused destruction of alpha(1)-antiproteinase and the HOCl-induced destruction of collagen, whereas they did not affect the elastase-induced destruction of collagen. Glutathione, tiopronin and cysteine almost completely decomposed both HOCl and H(2)O(2), while ambroxol up to 1 mM did not show a scavenging action on H(2)O(2). Ambroxol (1 to 100 microM) and 1 mM thiol compounds markedly inhibited the HOCl-induced alteration of elastase activity. Thiol compounds significantly attenuated the HOCl production caused by degraded immunoglobulin G-activated neutrophils. Ambroxol depressed superoxide and H(2)O(2) production induced by degraded immunoglobulin G-activated neutrophils and by lipopolysaccharide-activated alveolar macrophages in a dose-dependent manner. The results show that ambroxol may interfere with oxidative tissue damage and decrease proteolytic tissue destruction by attenuation of oxidative stress-induced inactivation of alpha(1)-antiproteinase through both decomposition of HOCl and inhibition of the respiratory burst in phagocytic cells.

Membrane orientation of carboxyl-terminal half P-glycoprotein: topogenesis of transmembrane segments.

Multiple topological orientations of the carboxyl-terminal half of P-glycoprotein have been observed. One orientation is consistent with the hydropathy-predicted model and contains six transmembrane (TM)-spanning regions. In another orientation, the cytoplasmic-predicted loop between TM8 and TM9 is extracellular and glycosylated. In support of this "alternative" topology, TM8 was previously established to function as a signal-anchor sequence to insert with its amino-terminal end in the cytoplasm and the carboxyl-terminal end in the extracytoplasmic space. However, it is unclear how downstream TM segments fold in the membrane when TM8 functions as a signal-anchor sequence. Here, we created several chimeric Pgp molecules to examine the membrane insertion of TM segments 9 and 10 using a cell-free system. We found that TM9 functions as a stop-transfer sequence when following the signal-anchor sequence, TM8. However, the stop-transfer activity of TM9 depends on the presence of TM10. In the absence of TM10, TM9 partially translocated across the membrane into the endoplasmic reticulum lumen. In contrast, TM9 efficiently stopped the translocation event of the nascent chain in the presence of TM10. Our results suggest that the membrane insertion of TM8 and TM9 establishes the extracellular loop between TM8 and TM9. Formation of this loop apparently involves the interactions between Pgp TM segments, which facilitate proper folding of the Pgp carboxyl-terminal half.

Mutational analysis of the RecJ exonuclease of Escherichia coli: identification of phosphoesterase motifs.

The recJ gene, identified in Escherichia coli, encodes a Mg(+2)-dependent 5'-to-3' exonuclease with high specificity for single-strand DNA. Genetic and biochemical experiments implicate RecJ exonuclease in homologous recombination, base excision, and methyl-directed mismatch repair. Genes encoding proteins with strong similarities to RecJ have been found in every eubacterial genome sequenced to date, with the exception of Mycoplasma and Mycobacterium tuberculosis. Multiple genes encoding proteins similar to RecJ are found in some eubacteria, including Bacillus and Helicobacter, and in the archaea. Among this divergent set of sequences, seven conserved motifs emerge. We demonstrate here that amino acids within six of these motifs are essential for both the biochemical and genetic functions of E. coli RecJ. These motifs may define interactions with Mg(2+) ions or substrate DNA. A large family of proteins more distantly related to RecJ is present in archaea, eubacteria, and eukaryotes, including a hypothetical protein in the MgPa adhesin operon of Mycoplasma, a domain of putative polyA polymerases in Synechocystis and Aquifex, PRUNE of Drosophila, and an exopolyphosphatase (PPX1) of Saccharomyces cereviseae. Because these six RecJ motifs are shared between exonucleases and exopolyphosphatases, they may constitute an ancient phosphoesterase domain now found in all kingdoms of life.

The inhibitory effect of ambroxol on respiratory burst, degranulation and cytosolic Ca2+ change in degraded immunoglobulin G-activated neutrophils.

Superoxide and H2O2 production by neutrophils stimulated by 0.5 mg/ml degraded immunoglobulin G (IgG) and 1 microM N-formyl-methionyl-leucyl-phenylalanine (fMLP) was inhibited by ambroxol in a dose-dependent fashion, and at the concentration of 100 microM, 43.3% to 64.3% of inhibitions were detected. The inhibitory effect of ambroxol on H2O2 production by neutrophils was greater than that on superoxide production. The production of nitrite by lipopolysaccharide-activated murine peritoneal macrophages was significantly attenuated by ambroxol in a dose-dependent fashion and NG-monomethyl-L-arginine (NMMA). Ambroxol decreased the release of myeloperoxidase and lysozyme evoked by 0.5 mg/ml degraded immunoglobulin G and 1 microM fMLP in a dose-dependent fashion, and at the concentration of 100 microM, 37.1% to 64.2% of inhibitions were observed. The stimulatory effect of phorbol 12-myristate 13-acetate (PMA) (0.1 microg/ml) on superoxide production and myeloperoxidase, which is inhibited by 100 nM staurosporine, was not affected by 100 microM ambroxol. Degraded immunoglobulin G (0.5 mg/ml) caused an immediate elevation of [Ca2+]i in fura-2 load neutrophils in 1.23 mM Ca2+-containing medium. Preincubation of neutrophils with 10 microM to 100 microM ambroxol, 5 mM EGTA and 100 microM verapamil depressed the elevation of [Ca2+]i elicited by 0.5 mg/ml degraded immunoglobulin G. In conclusion, the inhibitory action of ambroxol on stimulated neutrophil responses, including respiratory burst and lysosomal enzyme release, appears to be attributed to its depressant action on the activation process, including the change in intracellular Ca2+ level. in which the role of protein kinase C is uncertain.

Use of an antibody against the soluble interleukin 2 receptor alpha subunit can modulate the stability and biodistribution of interleukin-2.

The authors have previously reported that the soluble serum form of the alpha subunit of the IL-2 receptor (sIL-2Ralpha), whose natural half-life is approximately 40 min, survived much longer in the circulation when bound by a specific antibody. In the present study, the authors evaluated the extent to which sIL-2Ralpha protected IL-2 in freshly collected serum using biochemical analyses, and a functional CTLL-2 assay. In particular, sIL-2Ralpha protected IL-2 from forming complexes with alpha(2)-macroglobulin and from inactivation in vitro. In addition, the authors demonstrated that the anti-IL-2Ralpha monoclonal antibody 7G7/B6, which does not inhibit the binding of IL-2 to its binding site on sIL-2Ralpha, protected IL-2 from degradation and inactivation in vivo in the presence of sIL-2Ralpha. Both(125)I-labelled and unlabelled IL-2 were injected into mice preinjected with humanized anti-Tac (hTac) or 7G7/B6 and sIL-2Ralpha, or sIL-2Ralpha alone. Using size-exclusion HPLC, ELISA, and CTLL-2 cell proliferation assays, we observed that the presence of 7G7/B6 led to formation of complexes with sIL-2Ralpha and increased the serum levels of IL-2 more than 3- to 40-fold those of groups receiving IL-2 alone, sIL-2Ralpha, or hTac. Taken as a whole, these results suggest that the complex of 7G7/B6 and sIL-2Ralpha not only prolongs the survival of IL-2 in vivo, but also maintains the bioactivity of IL-2. The use of antibodies against endogenous soluble receptors could increase the in vivo survival of cytokines, protect their bioactivity and thereby facilitate their clinical use in the treatment of various malignancies and AIDS.