[Evaluation of the efficacy of urine-based lipoarabinomannan antigen test in the diagnosis of pulmonary tuberculosis].

Objective: To analyze the diagnostic efficacy of urinary lipoarabinomannan (LAM) antigen detection method in tuberculosis patients, and to provide an experimental basis for the clinical application of urinary LAM kit in China. Methods: From March to May 2023, 228 patients with lung diseases [134 male, 94 female, age 20-82 (44.8±16.7) years] were prospectively collected in Beijing Chest Hospital, Capital Medical University, including 143 pulmonary tuberculosis patients and 85 non-tuberculosis patients. Urine and sputum samples from patients were collected for traditional etiological detection and urinary LAM antigen detection. The screening results of each positive detection combination were analyzed, and the difference analysis and regression analysis were performed. Results: The detection sensitivity and specificity of the urinary LAM kit were 46.2% (95%CI: 37.9%-54.7%) and 96.5% (95%CI: 89.3%-99.1%), respectively, with an overall coincidence rate of 64.9%. The detection rate of LAM antigen detection and GeneXpert MTB/RIF (Xpert) combined (60.8%, 87/143) was significantly higher than that of Xpert alone (49.7%, 71/143), and the difference was statistically significant (P<0.05). The results of risk factor analysis showed that the risk of negative urinary LAM antigen test results increased significantly as the bacterial load decreased. Conclusions: Urine LAM antigen detection method has a high specificity and can be combined with traditional methods to effectively improve the detection rate. Urinary LAM antigen detection method still has limitations, such as the influence of bacterial load and the inability to distinguish nontuberculosis mycobacteria samples, which needs further experimental verification.

[Tolvaptan attenuates atrial remodeling in rats undergoing chronic intermittent hypoxia via miRNA-21].

Objective: To investigate the effects and potential mechanisms of tolvaptan on chronic intermittent hypoxia (CIH)-induced atrial remodeling in rats. Methods: A total of 45 Sprague-Dawley rats were divided into 3 groups by the random number table: control group, CIH group (6 h/d for 30 days), CIH plus tolvaptan group (8 mg·kg(-1)·d(-1) per gavage for 30 days). Echocardiography examination was performed after 30 days. Thereafter, 5 rats were randomly chosen for histology evaluation, 5 for molecular biological examinations and another 5 rats underwent isolated heart electrophysiology study in each group. Protein and mRNA expression levels of miRNA-21, Spry1, PTEN, ERK/p-ERK, MMP-9, PI3K, AKT/p-AKT were detected. Results: Compared to the rats in control group, rats in the CIH group showed higher atrial interstitial collagen deposition (P<0.001), increased atrial fibrillation inducibility (P=0.022). The results of immunohistochemistry staining showed that the mean optical density (MOD) of ERK, p-ERK and MMP-9 were significantly increased (all P<0.05), the MOD of Spry1 and PTEN were significantly decreased (both P<0.05), above changes could be significantly reversed by cotreatment with tolvaptan. No significant differences were detected in PI3K and AKT among the three groups (P>0.05). In addition, compared with rats in control group, mRNA levels of miRNA-21, MMP-9, PI3K, AKT, and protein levels of ERK, p-ERK, MMP-9 were significantly increased in CIH group(all P<0.05), whereas protein levels of Spry1, PI3K, p-AKT were significantly decreased (all P<0.05). Above changes could be significantly attenuated. Conclusions: CIH induces significant atrial remodeling in this rat model, which can be attenuated by tolvaptan possibly through modulating miRNA-21/Spry1/ERK/MMP-9 and miRNA-21/PTEN/PI3K/AKT signaling pathways.

Isolation and characterization of atrazine-degrading strain Shewanella sp. YJY4 from cornfield soil.

UNLABELLED: Atrazine has been used worldwide for over 50 years as a chemical herbicide. A strain of bacteria, YJY4 which utilizes atrazine as its sole nitrogen source for growth was isolated from agricultural black soil in northeastern China. 16S rDNA sequencing identified YJY4 as a Shewanella sp. PCR analysis and sequencing confirmed that YJY4 contained atrazine-degrading atzA, atzB and atzC genes. These genes revealed high similarity with those in Pseudomonas sp. ADP and Arthrobacter sp. TC1. The strain YJY4 was observed to degrade atrazine (100 mg l(-1) ) to cyanuric acid completely after 36 h. To the best of our knowledge, YJY4 was the first reported Shewanella sp. to grow in pure culture with atrazine serving as a sole source of nitrogen. Therefore, YJY4 may help with atrazine biodegradation and may become an abundant resource of atrazine degradation strains. SIGNIFICANCE AND IMPACT OF STUDY: A new isolated strain, YJY4 showed high atrazine-degrading ability, being able to degrade 100 mg l(-1) atrazine completely in 36 h. Strain YJY4 was identified as Shewanella sp. and contained atrazine-degrading atzA, atzB and atzC genes. This study examined the degradation mechanism and metabolic ability of this strain and for the bioremediation of contaminated environments, provides more strain selection and determines the strain of atrazine bioremediation potential.

[Chronic hypoxia increases intracellular Ca(2+) concentration and augments proliferation by enhancing store-operated Ca(2+) entry in pulmonary arterial smooth muscle cells].

OBJECTIVE: To determine whether store-operated Ca(2+) entry (SOCE) is involved in chronic hypoxia-induced alteration of intracellular Ca(2+) concentration ([Ca(2+) ]i) and proliferation in pulmonary arterial smooth muscle cells (PASMC). METHODS: Rat PASMCs were cultured and treated in normoxia (21%O2) or hypoxia (4%O2) condition. The proliferation of PASMC was detected by cell counting kit-8 (CCK-8) assay. [Ca(2+) ]i, SOCE and the effects of store-operated Ca(2+) channel (SOCC) inhibitors, SKF96365 and NiCl2, on SOCE in hypoxic PASMCs were tested by InCyte [Ca(2+) ]i measurement system. RESULTS: Hypoxia for 24-60 h augmented PASMC proliferation (1.12±0.09 vs 0.71±0.05, P<0.05) and [Ca(2+) ]i [(214.8 ± 20.4) nmol/L vs (115.2±13.2) nmol/L, P<0.05] in a time-dependent manner with the maximum effect at 60 h. Perfusion of Ca(2+) -free Krebs solution containing nifedipine (5 µmol/L), cyclopiazonic acid (CPA, 10 µmol/L) in PASMCs caused a small transient increase of [Ca(2+) ]i with peak [Ca(2+) ]i (113.3±49.3) nmol/L.Chronic hypoxia (4% O2, 60 h) enhanced [Ca(2+) ]i level with peak value of (193.2±22.7) nmol/L (P<0.05) in PASMC.After restoration of extracellular Ca(2+) , CPA caused marked increase of [Ca(2+) ]i with peak value of (328.0 ±56.7) nmol/L.Chronic hypoxia strengthened CPA-induced increase of [Ca(2+) ]i with peak value of (526.0±33.7) nmol/L (P<0.05) in PASMCs.Either SKF96365 50 µmol/L or NiCl2 500 µmol/L distinctly attenuated CPA-induced enhancement of [Ca(2+) ]i, the peak value of which dropped from (526.0±33.7) nmol/L to (170.4±26.4) nmol/L (P<0.05) or (177.4±45.9) nmol/L (P<0.05) respectively. CONCLUSION: Chronic hypoxia boosts the release of Ca(2+) from sarcoplasmic reticulum and promotes the activity of SOCC and SOCE, leading to [Ca(2+) ]i elevation and proliferation of rat PASMCs.

Comparison of effects of devascularization versus shunt on patients with portal hypertension: a meta-analysis.

OBJECTIVE: To systematically evaluate the effectiveness of devascularization and shunt on patients with portal hypertension. METHODS: Relevant studies compared devascularization and shunt for the treatment of portal hypertension were identified searching the PubMed, Embase, Elsevier, CNKI (China National Knowledge Infrastructure) database and Cochrane Trial Register searches until December 2013. Data of interest for devascularization and shunt including postoperative recurrent bleeding, postoperative hepatic encephalopathy,ascites, operative mortality rate, and long term survival rate were subjected to meta-analysis. RESULTS: Eleven studies were included in the study, the results of the meta-analysis showed that all eleven clinical studies demonstrated a significantly higher postoperative recurrent bleeding rate with devascularization group than with shunt group (Odds Ratio =2.14, 95% CI =(1.42, 3.21), P = 0.0003),the rate of hepatic encephalopathy in the devascularization group was significantly lower compared with the shunt group (Odds Ratio =0.56, 95% CI =(0.38, 0.82), P = 0.003); Our meta-analysis of three clinical studies revealed that the reduction of ascites in the devascularization group was significantly less than the shunt groups (Odds Ratio =0.48, 95% CI =(0.26, 0.89), P = 0.02), the operative mortality rate was not significantly different between the devascularization group than for shunt group (Odds Ratio =1.54, 95% CI = (0.91,2.63), P = 0.11). And the long-term survival rate was not significantly different between the devascularization and shunt groups (Odds=1.13, ratio, 95% CI =(0.64, 1.99), P = 0.68). CONCLUSIONS: Devascularization and shunt have different advantages and disadvantages respectively which reflected in postoperative complications and long term survival rate.

Transfer of Ser7 phosphorylated CENP-A from centromere to midbody during mitosis in MCF-7 cells.

Serine 7 of centromere protein A (CENP-A) is a very important mitosis-specific phosphorylation site. In this study, we demonstrate the subcellular distribution of Ser7 phosphorylated CENP-A during mitosis in MCF-7 cells. The Ser7 phosphorylation of CENP-A was observed beginning at prophase at centromeres. Upon progression of mitosis, the fluorescence signals emerged in the central region of the metaphase plate and were maintained until anaphase at centromeres. At late anaphase, the fluorescence signals moved to the midzone gradually and transferred from the centromere to the midbody completely at telophase. They were compacted into the centre of the midbody in a thin cylinder consisting of a sandglass-like "mitotic machine" with microtubules and condensed chromosome. We also found that Ser10 phosphorylated H3 and Thr11 phosphorylated H3 were co-localized at the midbody in two bell-like symmetrical bodies with Ser7 phosphorylated CENP-A during the terminal stage of cytokinesis. Midbody isolation and immunoblotting experiments also indicated that Ser7 phosphorylated CENP-A are components of the midbody. These findings suggest that Ser7 phosphorylated CENP-A acts as a chromosomal passenger protein and may play an important role in cytokinesis.

Neurofibromatosis type-1 with retroperitoneal stromal tumour: one case report.

BACKGROUND: Neurofibromatosis with gastrointestinal stromal tumours have been reported several times, while neurofibromatosis with retroperitoneal stromal tumours are very rare. CASE DESCRIPTION: We report the case of a 44-year-old man with a long history of neurofibromatosis. He complained of severe constipation and left leg pain. The patient's examination showed prominent peripheral cutaneous neurofibromas mainly in the belly and limbs, especially a huge mass in his abdomen, no less than ten café-au-lait spots, four Lisch nodules of the iris. Computed tomography and magnetic resonance imaging revealed a round and lobular mass in the retroperitoneal space. It was a well-circumscribed, hypervascular mass with cystic necrosis. A surgical resection was performed, and pathology and immunohistochemistry findings were consistent with stromal tumour. The c-kit gene and platelet-derived growth factor receptor-α gene mutations are not observed in the specimen. CONCLUSIONS: Neurofibromatosis with retroperitoneal stromal tumour is very rare, and radiological, pathological and immunohistochemical examination may identify it. Surgical resection may be the unique method of cure for it.

Molecular mechanisms of MCM3AP-AS1 targeted the regulation of miR-708-5p on cell proliferation and apoptosis in gastric cancer cells.

The article "Molecular mechanisms of MCM3AP-AS1 targeted the regulation of miR-708-5p on cell proliferation and apoptosis in gastric cancer cells, by H. Wang, T. Xu, L. Wu, H.-L. Xu, R.-M. Liu, published in Eur Rev Med Pharmacol Sci 2020; 24 (5): 2452-2461-DOI: 10.26355/eurrev_202003_20512-PMID: 32196596" has been withdrawn from the authors due to the discovery of new results. The authors decided to improve them further. The Publisher apologizes for any inconvenience this may cause. https://www.europeanreview.org/article/20512.

Molecular mechanisms of MCM3AP-AS1 targeted the regulation of miR-708-5p on cell proliferation and apoptosis in gastric cancer cells.

OBJECTIVE: Gastric cancer (GC) is a common malignancy of the digestive tract. Accumulated studies proved that long non-coding RNA MCM3AP-AS1 (MCM3AP-AS1) modified the mechanism of the progression of GC. However, the molecular mechanism is still greater elusive. Hence, we aimed to explore the molecular mechanism of MCM3AP-AS1 targeting the regulation of microRNA-708-5p on cell proliferation and apoptosis in GC cells. MATERIALS AND METHODS: The expression levels of MCM3AP-AS1 (MCM3AP antisense RNA 1) in gastric mucosal cells GES-1 and gastric cancer cell lines of MGc-803 and SGC-7901 cells were detected by qRT-PCR. Moreover, the protein levels of Cyclin D1, P21, Bax and Bcl-2 in MGc-803 and SGC-7901 cells after transfection were detected by Western blot. MTT assay was performed to detect cell proliferation and flow cytometry was carried out to determine GC cell apoptosis in vitro. In the endpoint, the targeting relationship between MCM3AP-AS1 and microRNA-708-5p was detected by Dual-Luciferase reporter assay. RESULTS: The level of MCM3AP-AS1 was significantly promoted in GC cell lines. Knockdown of MCM3AP-AS1 curbed cell proliferation and enhanced apoptosis in MGc-803 and SGC-7901 cells. Furthermore, the effect of the downregulation of MCM3AP-AS1 on cell proliferation and apoptosis was reversed by knockdown of miR-708-5p, which was targeted by MCM3AP-AS1 in vitro. CONCLUSIONS: MCM3AP-AS1 regulates the proliferation and apoptosis of gastric cancer cells by targeting the expression of microRNA-708-5p. The study may be useful to the therapy target of human GC.

Phase transitions in the classical exchange-anisotropic Kitaev-Heisenberg model.

The Kitaev model on the honeycomb lattice has been receiving substantial attention due to the discovery of quantum spin liquid state associated with this model. Consequently, its classical partners such as the Kitaev-Heisenberg (KH) model and associated phase transitions become concerned. Specifically, an intermediate Kosterlitz-Thouless (KT) phase engaged in the transition from the high-temperature (T) disordered state to the low-T sixfold degenerate state is predicted in the isotropic KH model [Phys. Rev. Lett. 109, 187201 (2012)10.1103/PhysRevLett.109.187201], but so far no sufficient experimental proof has been reported. In this work, we consider an essential extension of this KH model on the honeycomb lattice by including the Kitaev exchange anisotropy that is non-negligible in realistic materials. The associated phase transitions are thus investigated using the Monte Carlo simulations. It is found that such an anisotropy will result in a degradation of the sixfold degeneracy of the ground state in the isotropic KH model down to the fourfold or twofold degenerate ground state, and the finite-T phase transitions will also be modified remarkably. Interestingly, the intermediate KT phase can be suppressed by this Kitaev exchange anisotropy. This work thus provides a more realistic description of the physics ingredient with the KH model and presents a possible explanation on absence of the intermediate phase in real materials where the Kitaev exchange anisotropy can be more or less available.

Synthetic chimeric nucleases function for efficient genome editing.

CRISPR-Cas systems have revolutionized genome editing across a broad range of biotechnological endeavors. Many CRISPR-Cas nucleases have been identified and engineered for improved capabilities. Given the modular structure of such enzymes, we hypothesized that engineering chimeric sequences would generate non-natural variants that span the kinetic parameter landscape, and thus provide for the rapid selection of nucleases fit for a particular editing system. Here, we design a chimeric Cas12a-type library with approximately 560 synthetic chimeras, and select several functional variants. We demonstrate that certain nuclease domains can be recombined across distantly related nuclease templates to produce variants that function in bacteria, yeast, and human cell lines. We further characterize selected chimeric nucleases and find that they have different protospacer adjacent motif (PAM) preferences and the M44 chimera has higher specificity relative to wild-type (WT) sequences. This demonstration opens up the possibility of generating nuclease sequences with implications across biotechnology.

Magnetic impurity doping induced spin-glass state and short-range zigzag order in the honeycomb iridate Na2IrO3.

Based on the modified Heisenberg-Kitaev model, the effects of magnetic substitution on the magnetic properties of the honeycomb-lattice iridate [Formula: see text] [Formula: see text] are studied using Monte Carlo simulations. It is observed that the long-range zigzag state of the original system is rather fragile and can be replaced by a spin-glass state even for small substitution, well consistent with the experimental observation in Ru-substituted samples (Mehlawat et al 2015 Phys. Rev. B 92 134412). Both the disordered Heisenberg and Kitaev interactions caused by the magnetic ion-doping are suggested to be responsible for the magnetic phase transitions in the system. More interestingly, a short-range zigzag order is suggested to survive above the freezing temperature even at high magnetic impurity doping levels.

Ashkin-Teller criticality and weak first-order behavior of the phase transition to a fourfold degenerate state in two-dimensional frustrated Ising antiferromagnets.

We study the thermal phase transition of the fourfold degenerate phases (the plaquette and single-stripe states) in the two-dimensional frustrated Ising model on the Shastry-Sutherland lattice using Monte Carlo simulations. The critical Ashkin-Teller-like behavior is identified both in the plaquette phase region and the single-stripe phase region. The four-state Potts critical end points differentiating the continuous transitions from the first-order ones are estimated based on finite-size-scaling analyses. Furthermore, a similar behavior of the transition to the fourfold single-stripe phase is also observed in the anisotropic triangular Ising model. Thus, this work clearly demonstrates that the transitions to the fourfold degenerate states of two-dimensional Ising antiferromagnets exhibit similar transition behavior.

Dynamic distribution of Thr3-phosphorylated histone H3 in CHO cells in mitosis.

The phosphorylation of histone H3 at Ser10, Ser28, Thr11 and Thr3 of the amino terminal has been proved related to mitosis of the mammalian cells. However, the function of the Thr3 phosphorylation of H3 remains unclear. In this study, indirect immunofluorescence labelling and laser confocal microscopy were used to examine the cellular dynamic distribution of Thr3-phosphorylated H3 at mitosis in CHO cells. The results showed that the Thr3 phosphorylation began at early prophase and spread throughout the chromosomes at late prophase. At metaphase, most of the Thr3-phosphorylated H3 was distributed along the entire chromosomal arms and maintained until early anaphase. During late anaphase and telophase, the fluorescent signal of Thr3-phosphorylated H3 disappeared from chromosomes. There was a precise spatial and temporal correlation between H3 phosphorylation of Thr3 and stages of chromatin condensation. The timing of Thr3 phosphorylation and dephosphorylation in mitosis were similar to that reported for Thr11 phosphorylation of H3. The Thr3-phosphorylated H3 localized along the arms of chromosomes during metaphase and early anaphase. It was different from the Ser10-phosphorylated H3, which localized at telomere regions, and Thr11-phosphorylated H3, which localized at centromeres. The results suggest that the Thr3 phosphorylation of histone H3 may play a specific role, which is different from Ser10 phosphorylation and Thr11 phosphorylation in mitosis.

Role of further-neighbor interactions in modulating the critical behavior of the Ising model with frustration.

In this work, we investigate the phase transitions and critical behaviors of the frustrated J(1)-J(2)-J(3) Ising model on the square lattice using Monte Carlo simulations, and particular attention goes to the effect of the second-next-nearest-neighbor interaction J(3) on the phase transition from a disordered state to the single stripe antiferromagnetic state. A continuous Ashkin-Teller-like transition behavior in a certain range of J(3) is identified, while the four-state Potts-critical end point [J(3)/J(1)](C) is estimated based on the analytic method reported in earlier work [Jin, Sen, and Sandvik, Phys. Rev. Lett. 108, 045702 (2012)]. It is suggested that the interaction J(3) can tune the transition temperature and in turn modulate the critical behaviors of the frustrated model. Furthermore, it is revealed that an antiferromagnetic J(3) can stabilize the staggered dimer state via a phase transition of strong first-order character.

Effects of cloned human leukocyte interferons in the human tumor stem cell assay.

Clonogenic tumor cells from fresh biopsies of human cancers were cultivated in vitro and tested for sensitivity by continuous exposure to pharmacologically achievable concentrations of either of two highly purified human leukocyte interferon subtypes (IFN-alpha A and IFN-alpha D) prepared by recombinant DNA methods. The interferons were compared on a weight basis at concentrations of 0.4 and 4.0 ng/ml (equivalent to 80 and 800 units of interferon activity for IFN-alpha A and 2.0 and 20 units for IFN-alpha D). Inhibition of tumor colony-forming units (50% of control or less) was observed in 38.1% of the 273 tumors tested against IFN-alpha A, and in 16% of the 71 tumors tested against IFN-alpha D. Of the tumor types with at least ten samples tested against IFN-alpha A, the percentage of cases exhibiting inhibition was as follows: melanoma (51.7%), lung cancer (50%), myeloma (33.4%), ovarian cancer (33.9%), sarcoma (33.3%), adenocarcinoma of unknown primary (30.4%), breast cancer (28%), acute leukemia (30.8%), and renal cancer (23%). More marked inhibition (30% of control or less) was observed in 18.7% of all tumors tested against IFN-alpha A. Of 60 melanomas tested, 18 (30%) exhibited marked in vitro inhibition of growth with IFN-alpha A. Although a smaller number of tumors (71) were tested against IFN-alpha D on a weight basis, it appeared, in general, to be slightly less active than IFN-alpha A (p less than 0.01), and only 8% of tumors tested exhibited marked inhibition over the same dosage range of interferon. Comparison of the dose-response curves for the 68 tumors tested simultaneously against both interferons did not reveal marked interpatient differences in the inhibition curves, although IFN-alpha D was slightly less active overall. Tumors exhibiting at least 50% inhibition of tumor colony formation also proved to be sensitive to a significantly larger number of cytotoxic drugs (tested simultaneously) than the tumors not inhibited with interferon (p less than 0.0001 for IFN-alpha A). We conclude that the in vitro clonogenic assay may aid in targeting tumor types most likely to exhibit interferon sensitivity and assist in case selection for entry into clinical trials with cloned interferons.

Adaptation to oxidative stress: quinone-mediated protection of signaling in rat lung epithelial L2 cells.

Cells can respond to a sublethal oxidative stress by up-regulating their intracellular glutathione (GSH) pool. Such increased GSH concentration is likely to be protective against further oxidative challenge, and, in fact, pre-exposure to low levels of oxidants confers increased cellular resistance to subsequent greater oxidative stress. Previously, we have shown that pretreatment of rat lung epithelial L2 cells with sublethal concentrations of tert-butylhydroquinone (TBHQ) increases intracellular GSH concentration in a concentration- and time-dependent manner. This increase resulted from up-regulation of both gamma-glutamyltranspeptidase (GGT) and gamma-glutamylcysteine synthetase (GCS). Therefore, we investigated whether such increased GSH concentration protected these cells against a subtle loss in function caused by a subsequent challenge with sublethal concentrations of tert-butyl hydroperoxide (tBOOH) (< or = 200 microM), mimicking a physiological oxidative stress. Activation of L2 cell purinoreceptors with 100 microM ADP caused an elevation of intracellular Ca2+. This response was suppressed by a brief pre-exposure to tBOOH. The inhibition, however, was alleviated dramatically by a 16-hr pretreatment with 50 microM TBHQ. The same TBHQ pretreatment also protected the cells from ATP-depletion induced by tBOOH. L-Buthionine S,R-sulfoximine (BSO), an irreversible inhibitor of GCS, prevented the increase in intracellular GSH and also completely removed the protection by TBHQ in maintaining the ATP level. Thus, pre-exposure to a sublethal level of TBHQ results in protection of cell functions from hydroperoxide toxicity. This protection appears to depend on alteration of the intracellular GSH pool, the modulation of which constitutes an adaptive response to oxidative stress.

Mechanisms of protection from menadione toxicity by 5,10-dihydroindeno[1,2,-b]indole in a sensitive and resistant mouse hepatocyte line.

Established cell lines derived from newborn livers of c14CoS/c14CoS and cch/cch mice have been shown to be genetically resistant (14CoS/14CoS cells) or susceptible (ch/ch cells) to menadione toxicity. These differences are due in part to relatively higher levels of reduced glutathione (GSH) and NAD(P)H:menadione oxidoreductase (NMO1) activity in the 14CoS/14CoS cells. The indolic membrane-stabilizing antioxidant 5,10-dihydroindeno[1,2-b]indole (DHII) was shown previously to protect against various hepatotoxicants in vivo and in primary rat hepatocytes. This report describes how the 14CoS/14CoS and ch/ch cell lines provide a valuable experimental system to distinguish the mechanism of chemoprotection by DHII from menadione toxicity. The addition of 25 microM DHII produced a time-dependent decrease in menadione-mediated cell death in 14CoS/14CoS cells, with little effect on ch/ch cell viability. The maximum protective effect occurred at 24 hr, although the concentration of DHII remained constant for 48 hr. The protective effect of DHII correlated with enhanced glutathione levels (234% increase at 24hr), as well as induction of four enzymes involved in the detoxification and excretion of menadione: NAD(P)H:menadione oxidoreductase (NMO1, quinone reductase), glutathione reductase, glutathione transferase (GST1A1), and UDP glucuronosyltransferase (UGT1*06), with 24-hr maximum induction of 707, 201, 171 and 198%, respectively. Other biotransformation enzymes not directly involved in menadione metabolism (glutathione peroxidase, cytochromes P4501A1 and P4501A2, copper-, zinc-dependent superoxide dismutase, and NADPH cytochrome c oxidoreductase) were not induced by DHII. Menadione-stimulated superoxide production was inhibited 50% by DHII only in 14CoS/14CoS cells, and the inhibition required 24-hr preincubation. Pretreatment with DHII also protected both cell types against the menadione-mediated depletion of GSH, and the increase in percent (oxidized glutathione GSSG), an indicator of oxidative stress. These results suggest that DHII does not protect against menadione toxicity by virtue of its antioxidant or membrane-stabilizing properties. Rather, it acts by inducing a protective enzyme profile that migates redox cycling and facilitates excretion of menadione.

Response of [Ah] battery genes to compounds that protect against menadione toxicity.

We have studied the response of genes in the dioxin-inducible [Ah] battery to three compounds that protect mouse hepatoma cells (Hepa-1c7c7 wild-type, wt) against menadione toxicity. Pretreatment of wt cells with 25 microM 5,10-dihydroindenol[1,2-b]indole (DHII), 25 microM tert-butylhydroquinone (tBHO) or 10 microM menadione itself, generated substantial protection against toxicity produced by subsequent menadione exposure. The gene response was examined in wt cells, and three mutant lines: CYP1A1 metabolism-deficient (c37 or P1-); nuclear translocation-impaired (c4 or nt-); and AHR-deficient (c2 or r-, containing < 10% of normal functional receptor levels). DHII treatment of wt cells for 12 hr markedly elevated the enzyme activities and mRNA levels of genes in the [Ah] battery: aryl hydrocarbon hydroxylase (Cyp1a1), NAD(P)H:menadione oxidoreductase (Nmol), cytosolic aldehyde dehydrogenase class 3 (Ahd4), and UDP-glucuronosyltransferase form 1*06 (Ugt1*06). Treatment of the c4 and c2 cells with DHII failed to induce mRNA levels of the genes, indicating that induction of the [Ah] gene battery by DHII is aromatic hydrocarbon receptor (AHR)-mediated. On the other hand, neither tBHO nor menadione caused increases in CYPlAl mRNA, but tBHQ significantly enhanced the NMO1, AHD4, and UGT1*06 mRNA levels in all three mutant cell lines. In conclusion, we expect one or more putative electrophile response elements (EpRE), previously found in the regulatory regions of the murine Nmol, Ahd4, and ugt1*06 genes, to be functional in responding to phenolic antioxidants.

Evaluation of new anthracycline analogs with the human tumor stem cell assay.

Ten anthracyclines, including doxorubicin (DX) and daunorubicin (DNR), and eight analogs with modifications in structure or stereochemistry of the aglycone and/or the aminosugar moiety were simultaneously tested in serial vitro titration studies against human adenocarcinomas in the human tumor stem cell assay. More than a two-log range in cytotoxicity of the various anthracyclines was observed with the tumors tested. Marked individual differences in sensitivity of specific tumors (breast, lung, peritoneal) were observed for the various analogs. By assessing average effects on survival of tumor colony-forming units (TCFU) in the tumors tested, the three compounds lacking the methoxyl group in position 4 of the aglycone (4-demethoxyDX, 4-demethoxy-4'-epiDX, 4-demethoxyDNR) all proved to be more cytotoxic than their parent compounds. Compounds modified in position 4' of the aminosugar were on average either as toxic (4' epiDX) or more toxic (4'-deoxyDX and 4'-0-methylDX) to TCFU than the parent compound DX. On average, 11-deoxyDX was less toxic than DX or the other eight anthracyclines tested. The results obtained are also in good general agreement with those previously reported for anthracyclines with human tumors in xenografts or cancer patients. These antitumor results viewed in concert with toxicology studies in normal mice (including evidence of a lack of cardiac toxicity) suggest that 4'deoxyDX may prove to be a clinically useful anthracycline analog. We also conclude that use of this clinically predictive in vitro soft agar assay provides a rapid and relatively inexpensive means of simultaneously testing a large number of analogs of a parent compound against a spectrum of human tumors.