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The global navigation satellite system (GNSS)-based multi-antenna attitude determination method has the advantages of a simple algorithm and no error accumulation with time in long endurance operation. However, it is sometimes difficult to simultaneous obtain the fixed solutions of all antennas in vehicle attitude determination. If float or incorrect fixed solutions are used, precision and reliability of attitude cannot be guaranteed. Given this fact, a baseline-constrained ambiguity function method (BCAFM) based on a self-built four GNSS antennas hardware platform is proposed. The coordinates obtained by BCAFM can replace the unreliable real-time kinematic (RTK) float or incorrect fixed solutions, so as to assist the direct method for attitude determination. In the proposed BCAFM, the baseline constraint is applied to improve search efficiency (searching time), and the ambiguity function value (AFV) formula is optimized to enhance the discrimination of true peak. The correctness of the proposed method is verified by vehicle attitude determination results and baseline length difference. Experimental results demonstrate that the function values of error peaks are reduced, and the only true peak can be identified accurately. The valid epoch proportion increases by 14.95% after true peak coordinates are used to replace the GNSS-RTK float or incorrect fixed solutions. The precision of the three attitude angles is 0.54°, 1.46°, and 1.15°, respectively. Meanwhile, the RMS of baseline length difference is 3.8 mm.

Tropospheric mapping function plays a vital role in the high precision Global Navigation Satellites Systems (GNSS) data processing for positioning. However, most mapping functions are derived under the assumption that atmospheric refractivity is spherically symmetric. In this paper, the pressure, temperature, and humidity fields of ERA5 data with the highest spatio-temporal resolution available from the European Centre for Medium-range Weather Forecast (ECMWF) were utilized to compute ray-traced delays by the software WHURT. Results reveal the universal asymmetry of the hydrostatic and wet tropospheric delays. To accurately represent these highly variable delays, a new mapping function that depends on elevation and azimuth angles—Tilting Mapping Function (TMF)—was applied. The basic idea is to assume an angle between the tropospheric zenith direction and the geometric zenith direction. Ray-traced delays served as the reference values. TMF coefficients were fitted by Levenberg–Marquardt nonlinear least-squares method. Comparisons demonstrate that the TMF can improve the MF-derived slant delay’s accuracy by 73%, 54% and 29% at the 5° elevation angle, against mapping functions based on the VMF3 concept, without, with a total and separate estimation of gradients, respectively. If all coefficients of a symmetric mapping function are determined together with gradients by a least-square fit at sufficient elevation angles, the accuracy is only 6% lower than TMF. By adopting the b and c coefficients of VMF3, TMF can keep its high accuracy with less computational cost, which could be meaningful for large-scale computing.

High-rate precise point positioning with a global positioning system (GPS) is recognized as a powerful tool for large earthquake monitoring. However, its ability to monitor subdaily subtle deformation is still limited by unmodeled errors, which primarily contain the common-mode error (CME) and multipath. In this study, a combination of modified principal component analysis and the sidereal filtering method is employed to separate and extract the CME and multipath from high-rate GPS displacements. The effectiveness of the proposed method is validated by processing 1 Hz GPS data at 131 static stations over 30 days, during which the 4 and 6 July 2019 Ridgecrest earthquakes occurred. The results show no obvious early afterslip within the first 5 h following the 6 July Mw 7.1 earthquake. The CME mainly consists of the first principal component and can be attributed to satellite orbit and clock errors. The proportion of the CME is higher than that of the multipath in the high-rate displacements, namely 36.7% versus 14.4%, 23.8% versus 18.1%, and 36.1% versus 12.5% for the east, north, and up components, respectively. By applying this method, the high-rate displacements can achieve precisions of 0.47, 0.50, and 1.60 cm in the three components. The results of the rapid fault-slip inversions indicate that the PPP fixed solution augmented with spatiotemporal filtering can improve fault-slip inversion and moment magnitude estimation and may lead to new findings for geophysics.

The global navigation satellite system (GNSS)-based attitude determination system has attracted more and more attention with the advantages of having simplified algorithms, a low price and errors that do not accumulate over time. However, GNSS signals may have poor quality or lose lock in some epochs with the influence of signal fading and the multipath effect. When the direct attitude determination method is applied, the primary baseline may not be available (ambiguity is not fixed), leading to the inability of attitude determination. With the gradual popularization of low-cost receivers, making full use of spatial redundancy information of multiple antennas brings new ideas to the GNSS-based attitude determination method. In this paper, an attitude angle conversion algorithm, selecting an arbitrary baseline as the primary baseline, is derived. A multi-antenna attitude determination method based on primary baseline switching is proposed, which is performed on a self-designed embedded software and hardware platform. The proposed method can increase the valid epoch proportion and attitude information. In the land vehicle test, reference results output from a high-accuracy integrated navigation system were used to evaluate the accuracy and reliability. The results indicate that the proposed method is correct and feasible. The valid epoch proportion is increased by 16.2%, which can effectively improve the availability of attitude determination. The RMS of the heading, pitch and roll angles are 0.52°, 1.25° and 1.16°.

Global pressure and temperature 2 wet (GPT2w) is an empirical model providing the mean values plus annual and semiannual amplitudes of weighted mean temperature (Tm), which makes it a widely used tool in converting zenith wet delay (ZWD) to precipitable water vapor (PWV) in GNSS meteorology. The model meets the needs of real-time Tm anywhere in the world without relying on any other meteorological observations compared with traditional Tm calculation methods. It outperforms the other empirical Tm models released in recent years. Due to the lack of the Tm vertical adjustment in the model, the accuracy of Tm estimated by the model is subject to certain constraints, especially at sites which have large altitude differences compared with the GPT2w grid points. We explored the Tm lapse rate for the vertical adjustment using 10 years of 37 monthly mean pressure level data from the European Center for Medium-Range Weather Forecasts (ECMWF) and extended the GPT2w model to a new one called the GPT2wh model. Three schemes with different height ranges were established to fit the Tm lapse rate, and the most appropriate scheme was selected by adopting the goodness of fit measures, including the coefficient of determination (R-squared) and the root mean square error (RMSE). In addition to the mean value, annual and semiannual amplitudes for Tm lapse rate on a regular 1° grid were determined and stored in the GPT2wh model. The performance of the new model was assessed against the GPT2w model using different data sources in 2011, i.e., the ECMWF data and globally distributed radiosonde data. The numerical results show that the GPT2wh model outperforms the GPT2w model with an improved RMSE of 7.36/5.00/2.45/1.37/0.51/0.03 K at different height levels in the ECMWF comparison. In comparison with the radiosonde data, the mean RMSE of the GPT2wh model improves by 0.33 K from 4.16 to 3.83 K, i.e., an approximately 8% improvement against the GPT2w model. The impact of Tm on GNSS-PWV was analyzed, showing that the GPT2wh model can effectively improve the accuracy of the converted PWV.

The integrity monitoring algorithm based on pseudorange observations has been widely used outdoors and plays an important role in ensuring the reliability of positioning. However, pseudorange observations are greatly affected by the error sources such as multipath, clock drift, and noise in indoor pseudolite system, thus the pseudorange observations cannot be applied to high-precision indoor positioning. In general, double differenced (DD) carrier phase observations are used to obtain a high-precision indoor positioning result. What&rsquo, s more, the carrier phase-based integrity monitoring (CRAIM) algorithm is applied to identify and exclude potential faults of the pseudolites. In this article, a holistic method is proposed to ensure the accuracy and reliability of positioning results. Firstly, if the reference pseudolite operates normally, extended Kalman filter is used for parameter estimation on the premise that the number of common pseudolites meets positioning requirements. Secondly, the innovation sequence in the Kalman filter is applied to construct test statistics and the corresponding threshold is determined from the Chi distribution with a given probability of false alert. The pseudolitehorizontal protection level (HPL) is calculated by the threshold and a prior probability of missed detection. Finally, compared the test statistics with the threshold to exclude the faultypseudolite for the reliability of positioning. The experiment results show that the proposed method improves the accuracy and stability of the results through faults detection and exclusion. This method ensures accuracies at the centimeter level for dynamic experiments and millimeter levels for static ones.

Ultra-wideband (UWB) technology is suitable for indoor positioning owing to its high resolution and penetration. However, the current UWB positioning methods not only fail to fully analyze errors, but do not have the ability to eliminate gross and large random errors. In this article, the errors of UWB indoor positioning are analyzed comprehensively, and the basic function model is given. An indoor positioning method based on a double difference UWB with ranging observations is proposed and realized. In the proposed method, two UWB rover stations and a common base station are introduced, and the known baseline length between two rovers is used as the constraint condition for quality control. The observations and coordinate estimations are constrained by the prior and posteriori, respectively, and the weight of ranging observations with large residuals is reduced. Two groups of static experiments are designed. After adopting the proposed method, the plane error of one rover is 3.4 cm and 2.1 cm, and plane error of another rover is 3.3 cm and 2.0 cm, respectively. The positioning precision is improved by more than 80% compared with the traditional method. In the dynamic experiment, the coordinates of the starting and ending point obtained by the proposed method are basically consistent with the truth value, and the positioning results are close to the reference trajectory. The experimental results show that the proposed method can eliminate systematic and large random errors and improve the positioning precision effectively.

The indoor pseudolite (PL) code-based solution is not sufficiently precise, and conducting prolonged static carrier phase observations is impractical due to the static PL geometric distribution. Thus, a high-precision indoor PL positioning, which is generally based on the known point initialization (KPI) method and adopts the extended Kalman filter (EKF), is used to obtain the PL float ambiguity solution. However, the first-order linear truncation error of the EKF cannot be neglected because the indoor space is small, and its performance is quite dependent on the accuracy of the initial coordinates in the KPI. In this study, a well-suited nonlinear parameter estimation method, which is expected to have high precision and low dependence on the initial coordinate values, namely the unscented Kalman filter (UKF), is introduced and applied in PL positioning. Based on the relative positioning model and the KPI method, the positioning performance of the EKF and UKF under code-based differential pseudolite (DPL) positioning and phase-based real-time kinematic (RTK) positioning modes is compared and analyzed. Numerical results indicate that the computational efficiencies of the EKF and UKF are of the same level, though the former is slightly superior to the latter. In terms of the DPL positioning results, the precision of the UKF is higher with a decimeter-level improvement compared with that of the EKF. The dependence on the accuracy of the initial coordinates of UKF is reduced to some extent, which makes it a convenient technique, especially in the PL ambiguity resolution of the RTK positioning with fast convergence speed. The UKF outperforms the EKF and is more practicable in indoor PL positioning. The UKF can also achieve a decimeter-level positioning precision in DPL and a centimeter-level positioning precision in RTK.

Water vapor is an important part in the atmosphere, but its spatial and temporal distribution is difficult to detect. Global Positioning System (GPS) water vapor tomography, which can sense three-dimensional water vapor distribution, has been developed as a research point in the fields of GPS meteorology. In this paper, a new water vapor tomography method based on a genetic algorithm (GA) is proposed to overcome the ill-conditioned problem. By using the proposed approach, it is not necessary to perform the matrix inversion process, and the water vapor tomography is no longer dependent on excessive constraints, priori information and external data, which give rise to many limitations and difficulties. Experiments in Hong Kong under rainy and rainless conditions show a serious ill-conditioned problem in the tomographic matrix by grayscale and condition numbers. Numerical results indicate that the proposed method achieves high levels of agreement and internal/external accuracy with the GAMIT-estimated slant water vapor (SWV) as a reference. Comparative results of water vapor density (WVD) derived from radiosonde data reveal that the tomographic results based on the GA are in good agreement with that of radiosonde measurements. In comparison to the traditional Least squares method, a reliable tomographic result with high accuracy can be achieved by the GA without the restrictions mentioned-above. Furthermore, the tomographic results in a rainless scenario are better than those of a rainy scenario, and the reasons are discussed in detail.

The hybrid structures show excellent performance on vibration reduction for ship, aircraft and spacecraft designs. Meanwhile, the topology optimization is widely used for structure vibration reduction and weight control. The design of hybrid structures considering simultaneous materials selection and topology optimization are big challenges in theoretical study and engineering applications. In this paper, according to the proposed laminate component method (LCM) and solid isotropic microstructure with penalty (SIMP) method, the mathematical formulations are presented for concurrent materials selection and topology optimizations of hybrid structures. Thickness distributions of the plies in laminate components are defined as materials selection design variables by LCM method. Relative densities of elements in the components are defined as topology design variables by SIMP method. Design examples of hybrid 3-bar truss structures and hybrid floating raft with vibration reduction requirements verified the effectiveness of the presented optimization models.

The relationship among the lysyl oxidase (LOX) G473A single nucleotide polymorphism (SNP), cigarette smoking and lung, colorectal, colon and rectum cancer susceptibility was studied in 200 cases of lung cancer, 335 cases of colorectal cancer including 130 cases of colon cancer and 205 cases of rectum cancer, and 335 healthy people in Tangshan, China. Peripheral blood DNA samples were collected, DNA sequencing and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) performed, followed by multivariate logistic regression analysis. In comparison to LOX473GG genotype carriers, individuals with LOX473AA exhibited a higher susceptibility to lung, colon-rectum, colon, and rectum cancers with OR values amounting to 3.84-, 2.74-, 2.75-, and 2.74-fold of the control, respectively. In the LOX 473AA-positive population, females were more susceptible than males to carcinogenesis with OR values (female vs. male): 5.25 vs. 3.23, 2.29 vs. 1.51, 2.27 vs. 1.45, and 2.25 vs. 1.53, respectively, for lung, colon-rectum combined, colon, and rectum cancers. LOX G473A polymorphism apparently elevated human sensitivity to cigarette smoking carcinogens for eliciting cancers in the lung and colon only. Thus, LOX G473A polymorphism positively correlates with carcinogenesis and it may be used as an ideal intrinsic biomarker for prediction or diagnosis of carcinogenesis in humans.

Cadmium (Cd) inhalation can result in emphysema. Cd exposure of rat lung fibroblasts (RFL6) enhanced levels of metal scavenging thiols, e.g., metallothionein (MT) and glutathione (GSH), and the heavy chain of gamma-glutamylcysteine synthetase (gamma-GCS), a key enzyme for GSH biosynthesis, concomitant with downregulation of lysyl oxidase (LO), a copper-dependent enzyme for crosslinking collagen and elastin in the extracellular matrix (ECM). Cd downregulation of LO in treated cells was closely accompanied by suppression of synthesis of collagen, a major structure component of the lung ECM. Using rats intratracheally instilled with cadmium chloride (30 microg, once a week) as an animal model, we further demonstrated that although 2-week Cd instillation induced a non-significant change in the lung LO activity and collagen synthesis, 4- and 6-week Cd instillation resulted in a steady decrease in the lung LO and collagen expression. The lung MT and total GSH levels were both upregulated upon the long-term Cd exposure. Emphysematous lesions were generated in lungs of 6-week Cd-dosed rats. Increases of cellular thiols by transfection of cells with MT-II expression vectors or treatment of cells with GSH monoethyl ester, a GSH delivery system, markedly inhibited LO mRNA levels and catalytic activities in the cell model. Thus, Cd upregulation of cellular thiols may be a critical cellular event facilitating downregulation of LO, a potential mechanism for Cd-induced emphysema.

Lysyl oxidase (LO) stabilizes the extracellular matrix by cross-linking collagen and elastin. To assess the transcriptional regulation of LO, we cloned the 5′-flanking region with 3,979 bp of the rat LO gene. LO transcription started at multiple sites clustered at the region from –78 to –51 upstream of ATG. The downstream core promoter element functionally independent of the initiator predominantly activated the TATA-less LO gene. 5′ Deletion assays illustrated a sequence of 804 bp upstream of ATG sufficient for eliciting the maximal promoter activity and the region –709/–598 exhibiting strongly enhancing effects on the reporter gene expression in transiently transfected RFL6 cells. DNase I footprinting assays showed a protected pattern existing in the fragment –612/–580, which contains a nuclear factor I (NFI)-binding site at the region –594/–580 confirmed by electrophoretic mobility supershift assays. Mutations on this acting site decreased both NFI binding affinity in gel shift assays and stimulation of SV40 promoter activities in cells transfected with the NFI-binding site-SV40 promoter chimeric construct. Furthermore, at least two functional NFI-binding sites, including another one located at –147/–133, were identified in the LO promoter region –804/–1. Only NFI-A and NFI-B were expressed in rat lung fibroblasts, and their interaction with the LO gene was sensitively modulated by exogenous stimuli such as cigarette smoke condensate. In conclusion, the isolated rat LO gene promoter contains functionally independent initiator and downstream core promoter elements, and the conserved NFI-binding sites play a critical role in the LO gene activation.

To probe mechanisms of cadmium (Cd) damage to the lung extracellular matrix (ECM), we developed Cd-resistant (CdR) rat lung fibroblasts (RFL6) by incubation with graded concentrations of Cd. CdR cells downregulated lysyl oxidase (LO), a copper (Cu)-dependent enzyme essential for crosslinking of collagen and elastin in the ECM, in conjunction with upregulation of other Cu-binding proteins including Cu,Zn-superoxide dismutase (SOD1), copper chaperone for SOD1 (CCS1), metallothionein (MT), and Menkes P-type ATPase (ATP7A), a Cu transporter in the membrane of the Golgi apparatus, as well as gamma-glutamylcysteine synthetase (gamma-GCS), an enzyme for glutathione biosynthesis. Reduction and loss of cytoplasmic distribution of LO in CdR cells were accompanied by its dislocation with the Menkes P-type ATPase and the endoplasmic reticulum marker. CdR cells displayed a defect in LO catalytic activity but an enhancement in Cu,Zn-SOD catalytic activity consistent with the protein expression levels of these enzymes. Although long-term Cd exposure of cells enhanced the Menkes P-type ATPase protein expression, actually, it reduced Cu-dependent catalytic activity of this enzyme in parallel with the deficiency of LO. The low level of 64Cu bound to the LO fraction and the high level of 64Cu bound to the MT fraction provide direct evidence for limitation of Cu bioavailability for LO existing in the CdR cells. These results suggest that downregulation of LO is linked with upregulation of other Cu-binding proteins and with alteration in Cu homeostasis in the CdR phenotype.

Copper (Cu)-dependent lysyl oxidase (LO) catalyzes crosslinking of collagen and elastin stabilizing the extracellular matrix (ECM). Chronic inhalation of cadmium (Cd), a toxic metal, induces emphysema. To probe mechanisms of Cd injury to the lung, we developed Cd-resistant (CdR) cells from rat fetal lung fibroblasts (RFL6) by chronic exposure to CdCl(2) from 1 to 40 microM and further examined their expressions of LO, LO substrates, and Cu-scavenging thiols. Levels of cellular thiols, metallothionein, and glutathione in CdR cells were elevated to 13.0- and 3.2-fold of parental controls, respectively, whereas LO mRNA and protein levels were markedly reduced in these cells, with catalytic activity declining to only 16% of the parental control. A conspicuous 52 kDa species rather then the normal 50 kDa proenzyme appeared in the CdR cell extract but not in the conditioned medium, which was codistributed with the endoplasmic reticulum marker [DiOC5(3)] within the cell, implying the Cd-induced 52 kDa species as a product of an abnormal LO-processing defect in secretion. Addition of Cu into CdR cell cultures enhanced the expression of LO mRNA, protein and catalytic activities reflecting limitation of Cu bioavailability for LO in these cells. With inhibition of LO, CdR cells also displayed downregulation of collagen and elastin, substrates of LO. Restoration of collagen synthesis by exposure of CdR cells to purified LO or Cu suggests that inhibition of LO and limitation of Cu cofactor by Cd, as key phenotype changes, accelerated collagen and elastin damage, a critical event pertinent to emphysema pathogenesis.

Lysyl oxidase (LO) catalyzes crosslinking of collagen and elastin essential for maintaining the structural integrity of the lung extracellular matrix (ECM). To understand mechanisms of cigarette smoke (CS)-induced emphysema, we investigated effects of cigarette smoke condensate (CSC), the particulate matter of CS, on LO mRNA expression in cultured rat fetal lung fibroblasts (RFL6). Exposure of RFL6 cells to 0-120 microg CSC/ml for 24 h induced a dose-dependent inhibition of LO steady-state mRNAs, for example, reducing transcript levels to below 10% of the control in cells incubated with 80-120 microg CSC/ml. Nuclear run-on assays indicated a marked reduction in LO relative transcriptional rates amounting to 27.7% of the control in cells treated with 120 microg CSC/ml. The actinomycin D-chase assay showed that CSC enhanced the instability of LO transcripts. The t1/2 for LO mRNA decay was decreased from 24 h in the control to 4.5 h in cells treated with 120 microg CSC/ml. Moreover, 80-120 microg CSC/ml also inhibited LO promoter activity as revealed by suppression of reporter gene expression in cells transfected with LO promoter-luciferase vectors. Thus, inhibition of LO transcription initiation and enhancement of LO mRNA instability both contributed to downregulation of LO steady-state mRNA in CSC-treated cells. Note that inhibition of LO mRNA expression by CSC was closely accompanied by markedly decreased levels of transcripts of collagen type I and tropoelastin, two substrates of LO. Thus, transcriptional perturbation of LO and its substrates may be a critical mechanism for ECM damage in CS-induced emphysema.

Microtubule (MT) assembly in vitro is accompanied by hydrolysis of tubulin-bound GTP at E-site. Ni2+, a human carcinogen, has been shown to markedly perturb the MT system in cultured cells and enhance MT assembly in vitro. To further probe the mechanisms of such multiple Ni2+ damaging actions on MT, we have focused on dissecting the role of the Ni2+/GTP interaction in influencing MT assembly in vitro as monitored by a turbidity assay at A350 at 27 degrees C using purified bovine brain MT proteins containing 162 microM each of Mg2+ and EGTA. MT assembly was initiated by addition of GTP and progressed in a GTP dose-dependent manner. The minimal and optimal exogenous [GTP] required for MT assembly were 15.6 and 500 microM, respectively. Replacement of GTP (25-87%) with increasing [NiCl2] while keeping the sum of [GTP] and [Ni2+] constant at 500 microM enabled MT assembly to proceed with shortened "lags" but reaching the same maximum plateau levels or elongation rates as with 500 microM GTP only. However, in reactions with Ni2+ replacing >94% of GTP, marked inhibition of MT assembly (lower plateaus) occurred. Electron microscopic (EM) examinations showed that MT formed with high Ni2+ substitutions for GTP appeared shorter, more numerous, and resistant to Ca2+ disruption than those assembled with 500 microM GTP only. Notably, in the presence of 500 microM Ni2+ with no GTP added, no typical MT were observed under EM, despite increases in turbidity of the reaction. In addition, the critical concentration of MT proteins required for assembly was also considerably decreased under conditions of Ni2+ replacements of GTP. These results point to an important role of GTP/Ni2+ interaction in modulating the Ni2+ enhancement of MT assembly in vitro.

Lysyl oxidase (LO) plays a central role in the crosslinking of collagen and elastin in the extracellular matrix. Here we demonstrate that basic fibroblast growth factor (bFGF), a polypeptide which regulates proliferation, differentiation, and migration of a variety of cell types, is a substrate of LO. The oxidation of lysine residues in bFGF by LO resulted in the covalent crosslinking of bFGF monomers to form dimers and higher order oligomers and dramatically altered its biological properties. Both the mitogenic potential and the nuclear localization of bFGF were markedly inhibited in the Swiss 3T3 cells upon its oxidation by LO. NIH 3T3 IgBNM 6-1 cells (6-1 cells) overexpress bFGF which participates in an autocrine mechanism accounting for the transformation of these cells into a tumorigenic state. Exposure of the 6-1 cells to nanomolar concentrations of LO in culture oxidized lysine and generated crosslinkages in bFGF within the cell and markedly reduced proliferative rates. The lack of LO expression has been correlated with hyperproliferative cell growth, while this enzyme has been identified as a suppressor of ras-induced tumorigenesis. The present results illustrate a mechanism by which LO can depress normal and transformed cell growth.

A tobacco-specific carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), is believed to contribute to the cancer burden in cigarette smokers. To evaluate NNK effects on the expression of lysyl oxidase (LOX), a tumor suppressor, we examined this enzyme at various levels in NNK-treated rat fetal lung fibroblasts (RFL6). Exposure of cells to NNK reduced levels of steady-states LOX mRNA and new transcript synthesis. NNK inhibited all LOX protein species in a dose-dependent manner. Although 300 µM NNK markedly decreased the level in the 46 kDa preproenzyme, under same conditions, there was no detectable amounts of the 50 kDa proenzyme and the 32 kDa mature enzyme suggesting NNK perturbing the LOX protein processing to its mature form. Moreover, NNK also suppressed LOX activities in conditioned media of treated cells. At the promoter level, NNK enhanced methylation of CpG, but decreased acetylation of histone H3 at the core promoter region of the LOX gene. These results indicated that transcriptional and translational processes of LOX are major targets for NNK. Thus, inactivation of tumor suppressor gene LOX may play a critical role in NNK carcinogenesis.

Lysyl oxidase (LO) catalyzes crosslink of collagen, elastin, and histone H1, stabilizing the extracellular matrix and cell nucleus. This enzyme displays dual functions for tumorigenesis, i.e., as a tumor suppressor inactivating the ras oncogene and as a tumor promoter enhancing malignant cell metastasis. To elucidate LO transcriptional regulation, we have cloned the 804 base pair region upstream of the translation start site (ATG) of the rat LO gene with the maximal promoter activity. Computer analysis indicated that at least four hypoxia-response element (HRE) consensuses (5′-ACGTG-3′) exist in the cloned LO promoter. Treatment of rat lung fibroblasts (RFL6) with CoCl2 (Co, 10–100 μM), a chemical hypoxia reagent, enhanced LO mRNA expression and promoter activities. Overexpression of LO was associated with upregulation of hypoxia-inducible factor (HIF)-1α at mRNA levels in cobalt (Co)–treated cells. Thus, LO is a hypoxia-responsive gene. Dominant negative-HIF-1α inhibited LO promoter activities stimulated by Co. Electrophoretic mobility shift, oligonucleotide competition, and in vitro translated HIF-1α binding assays indicated that only one HRE mapped at −387/−383 relative to ATG was functionally active among four consensuses. Site-directed mutation of this HRE significantly diminished the Co-induced and LO promoter-directed expression of the reporter gene. Cadmium (Cd), an inducer of reactive oxygen species, inhibited HIF-1α mRNA expression and HIF-1α binding to the LO gene in Co-treated cells as revealed by RT-PCR and ChIP assays, respectively. Thus, modulation of the HRE activity by Co and Cd plays a critical role in LO gene transactivation.

To understand mechanisms for arsenic toxicity in the lung, we examined effects of sodium m-arsenite (As3+) on microtubule (MT) assembly in vitro (0–40 µM), in cultured rat lung fibroblasts (RFL6, 0–20 µM for 24 h) and in the rat animal model (intratracheal instillation of 2.02 mg As/kg body weight, once a week for 5 weeks). As3+ induced a dose-dependent disassembly of cellular MTs and enhancement of the free tubulin pool, initiating an autoregulation of tubulin synthesis manifest as inhibition of steady-state mRNA levels of βI-tubulin in dosed lung cells and tissues. Spindle MT injuries by As3+ were concomitant with chromosomal disorientations. As3+ reduced the binding to tubulin of [3H]N-ethylmaleimide (NEM), an -SH group reagent, resulting in inhibition of MT polymerization in vitro with bovine brain tubulins which was abolished by addition of dithiothreitol (DTT) suggesting As3+ action upon tubulin through -SH groups. In response to As3+, cells elevated cellular thiols such as metallothionein. Taxol, a tubulin polymerization agent, antagonized both As3+ and NEM induced MT depolymerization. MT–associated proteins (MAPs) essential for the MT stability were markedly suppressed in As3+-treated cells. Thus, tubulin sulfhydryls and MAPs are major molecular targets for As3+ damage to the lung triggering MT disassembly cascades.

To understand the mechanisms of toxicity of hexavalent chromate (K 2 CrO 4 , Cr 6+ ), the effects of this metal ion on the organization of microtubules (MTs) and microfilaments (MFs), DNA synthesis, cytoskeletal protein synthesis, cytoskeletal protein sulphhydryls (-SH groups), and cellular glutathione (GSH) levels in 3T3 cells were examined. Exposure of cells to Cr 6+ for 16 hr resulted in a dose-dependent inhibition of DNA synthesis with 50% inhibition occurring at 16.6 μ m . Treatment of cells with 3.13 μ m -Cr 6+ for 16 hr resulted in a slight cell retraction and, in some cells, MT bundling, without much effect on the morphology of dense MFs. At 25 μ m , Cr 6+ caused disruption of the cell sheet, depolymerization of MTs, particularly those in the peripheral areas, and redistribution of MFs, which assumed a smearing morphology. Exposure to 100 μ m -Cr 6+ induced severe thinning of MTs and loss of MFs. Although doses of 3.13 μ m -Cr 6+ or less slightly enhanced the level of cytoskeletal protein synthesis (e.g. 38% increase at 3.13 μ m -Cr 6+ ), Cr 6+ at 6.25 μ m or more produced a dose-dependent inhibition of cytoskeletal protein synthesis (50% inhibition at 11.25 μ m ). Exposure of cells to Cr 6+ for 16 hr resulted in a dose- and time-dependent increase of cellular GSH level. Furthermore, the elevated cellular GSH induced by Cr 6+ was diminished by treatment with buthionine sulphoximine (BSO), a potent inhibitor of GSH biosynthesis. In addition, depletion of GSH by BSO increased cell sensitivity to Cr 6+ insult and aggravated Cr 6+ -induced cytoskeletal perturbation. However, Cr 6+ treatment of cells did not significantly affect the amount of cytoskeletal protein sulphhydryls. These results suggest that cytoskeletal injury may be an important part of the mechanism for Cr 6+ toxicity. The cytoskeletal damage may result directly from the inhibition of cytoskeletal protein synthesis rather than from the interaction between Cr 6+ and cytoskeletal protein sulphhydryls.

Lysyl oxidase (LO), a copper-dependent enzyme, plays a critical role in the formation and repair of the extracellular matrix (ECM) by catalyzing the crosslinking of elastin and collagen. To better understand mechanisms of cigarette smoke (CS)-induced emphysema, we examined changes in LO and its substrates, i.e., elastin and collagen type I, the major components of cellular thiols, i.e., metallothionein (MT) and glutathione (GSH), and gamma-glutamylcysteine synthetase (gamma-GCS), a key enzyme for GSH biosynthesis, in cigarette smoke condensate (CSC)-treated rat fetal lung fibroblasts (RFL6). Exposure of RFL6 cells to CSC decreased levels of LO catalytic activity, mRNA, and protein, i.e., the 46 kDa preproenzyme, the 50 kDa proenzyme and the 32 kDa mature enzyme in a dose-dependent manner. In addition, CSC also inhibited the expression of collagen type I and elastin, substrates of LO and important components of the lung ECM. Meanwhile, cellular thiols including MT and GSH as well as gamma-GCS were markedly upregulated in CSC-treated cells. To evaluate modulation of LO expression by cellular thiols, we further examined the effect of increased levels of GSH on LO expression at protein and catalytic levels. Interestingly, exposure of cells to glutathione monoethyl ester, a GSH delivery system, effectively elevated cellular GSH levels and induced a dose-dependent decrease in levels of the protein species and catalytic activity of LO. These results suggest that upregulation by CSC of cellular thiols may play an important role in the downregulation of LO and subsequently destabilization of the lung ECM in CS-induced emphysema.

As an essential metal for cell metabolism, Mg 2+ is known to exert antagonism on Ni 2+ genotoxic and other effects. This study examined the influence of Mg 2+ on Ni 2+ -induced changes in microtubule (MT) assembly in vitro, cytoplasmic MT organization, cellular glutathione (GSH), and cytoskeletal and cytosolic protein sulfhydryls (PSH). As determined by a turbidity assay at 27°C, Ni 2+ enhanced the in vitro MT assembly in a Pipes buffer by shortening the initial lag (nucleation phase) and increasing the rate of polymerization with a higher final plateau. However, presence of 1 m M exogenous MgCl 2 abolished the Ni 2+ enhancing effect. Exposure of 3T3 cells to 2 m M NiCl 2 for 20 hr resulted in perinuclear bundling of MTs and decreases in cytoskeletal and cytosolic PSH and cellular GSH levels. However, coincubation of cells with MgCl 2 (1.25–20 m M ) added to the culture medium markedly diminished the Ni 2+ injury to MT organization. Under these conditions the Ni 2+ interference with PSH was blocked such that both the cytoskeletal and cytosolic PSH levels returned to the range of control cells without metal treatment. Treatment of cells with Mg 2+ (1.25–5 m M ) for 20 hr slightly increased, while with higher Mg 2+ doses (>10 m M ) decreased, cellular GSH content. Importantly, in Ni 2+ -treated cultures, addition of Mg 2+ (1.25–10 m M ) elevated GSH levels to ≥200% of that in cells treated with Ni 2+ alone. Furthermore, these Ni 2+ and Mg 2+ (1.25–10 m M ) treated cells actually maintained GSH levels which were essentially unchanged from the basal level of control cells with no metal treatment. Although Mg 2+ replacement of Ni 2+ bound to MT proteins could be an important mechanism, cellular GSH may also be a critical factor in Mg 2+ antagonism on Ni 2+ -enhanced MT assembly in view of the essential role of tubulin PSH in modulating MT assembly and, in turn, the GSH modulation of PSH.

To understand the mechanisms of Cd2+ and Ni2+ cytotoxicity, we have studied the effects of these two metal ions on the organization of cytoskeletal elements, microtubules (MT) and microfilaments (MF), cytoskeletal protein sulfhydryls and cellular glutathione (GSH) in cultured 3T3 cells. At a metal ion dose that caused 95% inhibition of DNA synthesis, Cd2+ (10 microM, 16 h exposure) induced MT depolymerization whereas Ni2+ (2 mM, 20 h exposure) elicited MT aggregation and bundling. Under these conditions, Cd2+ and Ni2+ also caused MF aggregation and redistribution. Furthermore, exposure of cells to Cd2+ resulted in a dose-dependent increase in cytoskeletal protein sulfhydryls and cellular GSH levels. In contrast, treatment of cells with Ni2+ resulted in a dose-dependent decrease in cytoskeletal protein sulfhydryls as well as cellular GSH content. Time course studies showed that cells exposed to 10 microM Cd2+ exhibited a biphasic response in regulating their cytoskeletal protein sulfhydryls and cellular GSH, e.g. an initial decrease followed by a steady recovery and overshooting upon prolonged incubation. However, restoration of cytoskeletal protein sulfhydryls occurred approximately 2 h after commencement of cellular GSH recovery in Cd(2+)-treated cells. These results suggest that cellular GSH may play an important role in regulating cytoskeletal protein sulfhydryls. On the other hand, decrease of cellular GSH induced by Ni2+ might facilitate oxidation of cytoskeletal protein sulfhydryls and formation of disulfide bonds between individual MT polymers which would favor MT aggregation in Ni(2+)-exposed cells. In addition, we also demonstrated that elevation of cellular GSH in Cd(2+)-treated cells probably resulted from new GSH synthesis.

Although the redox cycling of paraquat (PQ) and the resultant “activated oxygen” generation are important in toxicity development, the intracellular events leading to cell injury remain unclear. To understand the mechanism of PQ-induced cell injury, we have studied the effects of PQ on DNA synthesis, cell proliferation, the cytoskeletal organization, particularly microtubules (MT) and microfilaments (MF), and the synthesis and composition of cytoskeletal proteins in mouse 3T3 cells. PQ treatment produced a dose-dependent inhibition of DNA synthesis and cell growth. Exposure of cells to PQ (313 μ m , 20 hr) resulted in MT aggregation and bundling as well as MF redistribution in the perinuclear area as revealed by fluorescence microscopy. Although this PQ dose inhibited DNA synthesis by 95%, it caused only a 22% decrease in protein synthesis of the cytoskeletal fraction. Higher doses of PQ (1250 μ m , 20 hr) caused (a) dramatic thinning out and loss of MT and (b) marked loss of MF cables and the appearance of numerous pine needle-like structures much finer and shorter than normal MF. Under these conditions, the synthesis of cytoskeletal proteins was decreased by about 83%. Further analysis of the cytoskeletal fraction from PQ-treated cells by sodium dodecyl sulfate gel electrophoresis showed (a) that tubulin was greatly diminished, in agreement with microscopic observations; (b) that two new protein bands appeared; and (c) another protein band which was also reduced considerably. These results indicate that the PQ-induced dose-dependent cytoskeletal injury may be important to the mechanism of cytotoxicity of this herbicide.

To understand the mechanisms of toxicity of 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), we have studied their effects on the cytoskeletal organization, particularly microtubules (MT) and microfilaments (MF), DNA synthesis, and the synthesis and composition of cytoskeletal proteins in mouse 3T3 cells. Exposure of cells to 2,4-D or 2,4,5-T resulted in a dose-dependent inhibition of DNA synthesis; 50% inhibition occurred at 2.21 mM and 0.90 mM for 2,4-D and 2,4,5-T, respectively. Furthermore, a strong synergistic inhibition of DNA synthesis was produced by mixtures (each having a total concentration of 1.25 mM) of 2,4-D with 2,4,5-T. Similarly, 2,4,5-T is more potent than 2,4-D in causing cytoskeletal perturbation as revealed by fluorescence microscopy. Treatment of cells with 2,4-D (2.5 mM) or 2,4,5-T (1.25 mM) for 20 h resulted in severe MT aggregation and the appearance of large bundles, which were organized in a rope-like structure in the former and a dramatic octopus-like pattern in the latter. Further, MT bundling is particularly severe in the cell center. Under these conditions, marked changes in MF organization also occurred as evidenced by clustering and crisscrossing of MF in the perinuclear region. A 1:1 mixture (final = 1.25 mM) of 2,4-D and 2,4,5-T, a formulation equivalent to Agent Orange composition, also induced a dramatic perturbation to the organization of MT and MF, resulting in the formation of ring-like structures. MT bundling is still apparent, especially around the outer edge of the "rings." MF are localized predominantly along the cell periphery, where they appear to be aggregated tightly forming patches. Surprisingly, the synthesis and composition of cytoskeletal proteins, which are resistant to detergent extraction but released by CaCl2, are essentially unaffected by 2,4-D or 2,4,5-T. These results suggest that the dramatic perturbation of the cytoskeletal morphology caused by these herbicides probably only results from a structural reorganization and redistribution of MT and MF.