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.

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.

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.

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.

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.

Food restriction differentially affects mRNAs encoding the major anterior pituitary tropic hormones.

Chronic food restriction (FR) leads to adaptive cellular changes, some of which retard aging. Moreover, some of these changes occur within weeks after onset of FR. Because neuroendocrine mechanisms may mediate these effects, we measured the effect of FR on the messenger ribonucleicacids (mRNAs) encoding all of the tropic hormones of the anterior pituitary (AP). Slot blot and solution hybridization were conducted on AP ribonucleicacid (RNA) samples obtained at 0500 h (AM) and 1500 h (PM) from 3-month-old male Fischer 344 rats fed ad libitum (AL) or FR (60% of AL calories) since 6 weeks of age. PolyA RNA/microgram total RNA was similar in AL and FR rats, indicating that there was no overall effect of FR on mRNA levels. The level of proopiomelanocortin (POMC) mRNA was not reduced by FR when expressed per microgram of RNA or as total AP content. By contrast, the total AP content of the mRNAs encoding LH beta, FSH beta, TSH beta, GH, and PRL was markedly reduced by FR. When expressed per microgram of RNA, however, only GH (AM and PM), FSH beta (AM), TSH beta (PM), and PRL (PM) were reduced by FR. These results reveal that FR differentially affects pituitary tropic hormone mRNA levels within weeks after onset of FR, and are consistent with a role for neuroendocrine alterations in the initiation of adaptive cellular responses to FR.

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.

Hyperadrenocorticism and food restriction-induced life extension in the rat: evidence for divergent regulation of pituitary proopiomelanocortin RNA and adrenocorticotropic hormone biosynthesis.

The increased diurnal elevation of plasma corticosterone (B) induced by food restriction (FR) may play a role in the life span extension of FR. We investigated whether FR alters adrenocorticotropic hormone (ACTH) and proopiomelanocortin (POMC) mRNA levels in plasma and anterior pituitary (AP), since these molecules both regulate and can be suppressed by B. Measurements were made in 3-month-old male Fischer 344 rats that had been ad libitum (AL) or FR (60% of AL calories) since 6 weeks of age. Plasma B was 2-fold higher in FR rats in the PM samples, but did not differ in AM samples. By contrast, plasma ACTH did not differ in the PM samples of FR and AL rats and was 20% lower in AM samples (p < .05) of FR rats. AP content of ACTH was 50% lower in FR rats in both AM and PM samples (p < .01). In contrast, AP contents of POMC and mRNA, primary transcript, and processing intermediate were not reduced in FR rats, and PM content of POMC primary transcript was elevated in FR rats (p < .05). The reduced pituitary and plasma ACTH of FR rats may be the consequence of their elevated plasma B levels. This study also suggests that factors other than elevated ACTH account for FR-induced hyperadrenocorticism. These results also indicate that POMC mRNA and ACTH biosyntheses are differentially regulated in FR rats.

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.

Effects of protein kinase inhibitors on the stimulated neutrophil responses by degraded immunoglobulin G.

Effects of protein kinase C inhibitors, staurosporine and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine dihydrochloride and protein tyrosine kinase inhibitors, genistein, tyrphostin and 2,5-dimethylcinnamate on the neutrophil responses stimulated by immunoglobulin G (IgG), complement C5a or platelet-activating factor were studied. After receptor binding, the role of protein kinase C and protein tyrosine kinase in the stimulation of neutrophil responses, superoxide production and lysosomal enzyme release in degraded IgG-activated neutrophils may be similar to chemoattractant-stimulated cells. In contrast to complement C5a or platelet-activating factor, protein tyrosine kinase appears to play an important role in the regulation of intracellular Ca2+ mobilization in neutrophils activated by degraded IgG rather than by protein kinase C.

Similarities in the biodistribution of iodine-labeled anti-Tac single-chain disulfide-stabilized Fv fragment and anti-Tac disulfide-stabilized Fv fragment.

We evaluated the biodistribution and pharmacokinetics of two different iodine-labeled Fv fragments of anti-Tac monoclonal antibody (MAb) in normal and tumor-bearing nude mice. One was a disulfide-stabilized Fv fragment (dsFv), and the other was a single-chain disulfide-stabilized Fv fragment (scdsFv). The scdsFv is a newly developed type of Fv fragment superior to the dsFv in which the VH and VL are linked by covalent bonds through a spacer arm and by an internal disulfide bond. These modifications increase the yield of scdsFv. Both reagents recognize the alpha subunit of the interleukin-2 receptor (IL-2Ralpha). The biodistribution of the Fv fragments was evaluated in normal mice co-injected with 50 mg of L-lysine and in a no-lysine control group. Biodistribution was also evaluated in nude mice bearing subcutaneous tumor xenografts derived from IL-2Ralpha-positive ATAC4 cells and receptor-negative A431 cells. These mice were co-injected with 125I-labeled anti-Tac scdsFv (6 microCi/0.7 microg) and 131I-labeled anti-Tac dsFv (2 microCi/0.7 microg) or with 131I-labeled anti-Tac scdsFv (6 microCi/0.7 microg) and 125I-labeled anti-Tac dsFv (4 microCi/0.7 microg). The biodistribution of 125I-labeled anti-Tac scdsFv and 131I-labeled anti-Tac dsFv was very similar in all organs and the tumors. The renal uptake of both reagents was blocked effectively (<93%) and similarly by lysine. The scdsFv cleared slightly faster from the circulation than did the dsFv because there were more aggregates of dsFv than of scdsFv (3% vs. 1%, respectively). The scdsFv-to-dsFv ratio ranged from 0.79 to 1.20 in all organs at all time points we examined. In conclusion, the first biodistribution study of an scdsFv molecule shows that the scdsFv had a biodistribution very similar to that of the dsFv and seems to be a good alternative to the dsFv because of its higher production yield.

Cyproheptadine augmentation of haloperidol in chronic schizophrenic patients: a double-blind placebo-controlled study.

A 6 week double-blind placebo-controlled trial of cyproheptadine augmentation of ongoing haloperidol treatment was conducted in 40 chronic schizophrenic in-patients. Cyproheptadine augmentation, compared to administration of haloperidol with placebo, did not produce a statistically significant improvement in psychotic symptoms. Cyproheptadine augmentation caused significant reduction in the extrapyramidal symptoms, which supports the atypical profile of antipsychotics. As to the neuroendocrinological effect, cyproheptadine augmentation did not reduce the plasma prolactin level but did induce a decrease in the plasma cortisol level. Although long-term follow-up studies are needed to confirm the results, this study suggests that cyproheptadine augmentation may be effective in treating chronic schizophrenic patients who are intolerant of extrapyramidal side effects of conventional antipsychotics.

Farnesylcysteine methyltransferase activity and Ras protein expression in human stomach tumor tissue.

The processing pathway of G-proteins and Ras family proteins includes the isoprenylation of the cysteine residue, followed by proteolysis of three terminal residues and alpha-carboxyl methyl esterification of the cysteine residue. Farnesylcysteine methyltransferase (FCMT) activity is responsible for the methylation reaction which play a role in the membrane attachment of a variety of cellular proteins. Four kinds of Ras protein (c-Ha-ras, c-N-Ras, c-Ki-Ras, pan-Ras) expression were detected in adenocarcinoma of human tissue by immunohistochemical method, and hematoxylin and eosin staining. The level of Ras protein in human stomach tumor tissues was much higher than in normal and peritumoral regions of the same biopsy samples. The FCMT activities of each cellular fractions were high in mitochondrial fraction followed by microsomal fraction, whole homogenate and cytosolic fraction. The inhibitory effect on FCMT activity on stomach tumor tissue was determined after treatment with 0.25 microM of S-adenosyl-L-homocysteine. S-adenosyl-L-homocysteine inhibited FCMT activity from 11.2% to 30.5%. These results suggested that FCMT might be involved in Ras proteins activity.

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.

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.

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.

Pulmonary calcification and skin sensitivities to tuberculin, histoplasmin and coccidioidin among Polynesians.

Pulmonary calcifications are fairly common in Western Samoa, but they cannot be attributed entirely to tuberculosis, since they are also produced by certain systemic mycoses and parasitic infestations. Furthermore, in many tropical areas, skin sensitivity to tuberculin is often due to certain unidentified weak sensitizing agents, rather than to the usual virulent tubercle bacilli.Skin sensitivity to histoplasmin and coccidioidin is absent in both Western Samoa and the Tokelau Islands; consequently, pulmonary calcifications found in these places cannot be attributed to either histoplasmosis or coccidioidomycosis.Both Western Samoans and Tokelauans exhibit non-specific reactions to tuberculin throughout their lives, but these reactions are clearer and more distinct in groups aged 15 years or more.The prevalence of non-specific sensitivity to tuberculin should be investigated further.

Mechanism involved in generating the carboxyl-terminal half topology of P-glycoprotein.

P-Glycoprotein (Pgp) is a polytopic membrane protein that consists of a tandem repeat of a transmembrane (TM) domain followed by a nucleotide-binding domain. For the carboxyl-terminal half (C-half) of Pgp, at least three different topological orientations have been observed. One major difference between these topologies is reflected in the membrane insertion property of TM8, which is predicted to (1) function as a stop-transfer sequence, (2) lack stop-transfer activity, or (3) function as a signal-anchor sequence. To understand the mechanism involved in generating multiple topological forms for the C-half of Pgp, we investigated the membrane insertion properties of TM segments using the Chinese hamster pgp1 Pgp as a model protein in a cell-free system. We found that TM8 alone or in the presence of TM7 functions as a signal-anchor sequence to insert into membranes with a cytoplasmic amino terminus and an extra-cytoplasmic carboxyl terminus. However, TM8 displayed stop-transfer activity when linked to the C-terminal end of the signal-anchor sequence, TM1. In addition, the membrane orientation of TM8 was found to be regulated by the charge distribution flanking TM8. Interestingly, we found that mammalian and wheat germ ribosomes differentially regulate the signal-anchor and stop-transfer properties of TM8. We conclude that the unique topogenic properties of TM8 direct the generation of multiple C-half topological orientations.

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.