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Bacterial fruit blotch (BFB) of cucurbits, caused by Acidovorax citrulli, is a major threat to commercial watermelon and melon production worldwide. At present, there are at least two genetically distinct sub-populations (group I and II) of A. citrulli that differ in host preference among cucurbit species and copper sensitivity. In this study, we analyzed the pilA gene sequences of 103 A. citrulli strains from China and other countries. Based on these data, we classified all tested A. citrulli strains into three types. The pilA-based type 1 strains in this study coincided with the previously established group I strains; while the type 2 strains coincided with group II strains. Ten strains that did not cluster with group I or II strains were classified into a new type, designated type 3. Based on differences in pilA sequences, we designed a multiplex PCR assay to distinguish the three A. citrulli pilus types. This multiplex PCR assay has proven to be viable for strain typing of 139 A. citrulli strains and for the detection of this pathogen in artificially inoculated seeds and leaves and naturally infected leaves and fruits. This assay proved to be rapid, accurate, reliable and applicable for early distinction of A. citrulli types associated with BFB epidemics. It may also inform the judicious and environmentally sound use of bactericides, especially copper-based compounds., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Purpose: Although vitamin D is reportedly associated with various cancers, the association between vitamin D and thyroid cancer is indefinite. We aimed to investigate whether this association applies to thyroid cancer (TC)., Methods: A total of 276 Chinese Han people were recruited in a current matched case-control study. Multivariable conditional logistic regression was computed to estimate the association between plasma 25(OH)D and papillary thyroid cancer (PTC). In addition, we searched relevant studies in PubMed and Web of Science databases before December 2017 to conduct a meta-analysis., Results: In our case-control study, plasma 25(OH)D concentration was inversely associated with PTC risk (highest tertile vs lowest tertile: adjusted OR = 0.25; 95% CI 0.10, 0.61; P trend  = 0.003). This association was independent of body mass index and physical activity (all adjusted P interaction  > 0.05). A total of 11 studies were included in the meta-analysis, among which ten studies have been published and one was our case-control study. Compared with 25(OH)D non-deficient group, the pooled OR of TC was 1.42 (95% CI 1.17, 1.73) in the deficient group. Similarly, blood 25(OH)D levels in patients with TC were tend to be lower than those in the controls (SMD = - 0.20, 95% CI - 0.36, - 0.03)., Conclusions: A high level of circulating 25(OH)D was associated with a decreased TC risk. This association has important significance in public health and should, therefore, be further studied.

Objective: To understand the epidemiological characteristics of influenza in Guangdong province, during the winter of 2017-2018, to provide evidence for response to the diversity of influenza, in different seasonal patterns. Methods: Data on weekly influenza surveillance from January 2016 to April 2018, were collected in Guangdong. Information on patients with Influenza-like illness (ILI), on influenza virus positive rates and on outbreaks during the winter of 2017 to 2018, was analyzed and compared with those in spring of 2016 and summer of 2017. χ (2) test and Fisher exact test were used. Results: In the above said winter, the average percentage of visits for ILI in 28 hospitals where sentinel surveillance program had been set, was 4.99% (157 235/3 149 656), which was above the level of the same period in the previous five years. The positive rates of influenza virus among samples collected from ILI outpatients and hospitalized cases under severe acute respiratory infection (SARI) were 28.33% (2 137/7 543) and 14.93% (256/1 715), with the proportions of B (Yamagata) as 70.43% (1 505/2 137) and 73.05% (187/256) respectively. A total of 257 influenza outbreaks were reported in the winter period, with 82.49% (212/257) occurred in elementary schools. Cases aged 6-14 years occurred in winter and spring appeared of having higher positive rate than those seen in summer ( P <0.05) whereas elderly cases aged 60 and above showed higher positive rate in summer than those in winter and spring two seasons ( P <0.05). Conclusions: Epidemiological characteristics of influenza appeared in Guangdong province, during the winter from 2017 to 2018, were correlated to Influenza B (Yamagata). Capacity on the implementation of surveillance programs and on the coverage of vaccination should be improved and increased in order to control influenza in different epidemic seasons, in Guangzhou.

Aim: To evaluate in vitro the physicochemical properties, cytotoxicity and calcium phosphate nucleation of an experimental light-curable pulp capping material composed of a resin with antibacterial monomer (MAE-DB) and Portland cement (PC)., Methodology: The experimental material was prepared by mixing PC with a resin containing MAE-DB at a 2 : 1 ratio. Cured pure resin containing MAE-DB served as control resin. ProRoot MTA and Dycal served as commercial controls. The depth of cure, degree of monomer conversion, water absorption and solubility of dry samples, calcium release, alkalinizing activity, calcium phosphate nucleation and the cytotoxicity of materials were evaluated. Statistical analysis was carried out using anova followed by Tukey's HSD test (equal variance assumed) or Tamhane test (equal variance not assumed) and independent-samples t-tests., Results: The experimental material had a cure depth of 1.19 mm, and the mean degree of monomer conversion was 70.93% immediately post-cure and 88.75% at 24 h post-cure. The water absorption of the experimental material was between those of MTA and Dycal, and its solubility was significantly less (P < 0.05) than that of Dycal and higher than that of MTA. The experimental material exhibited continuous calcium release and an alkalinizing power between those of MTA and Dycal throughout the test period. Freshly set experimental material, control resin and all 24-h set materials had acceptable cytotoxicity. The experimental material, MTA and Dycal all exhibited the formation of apatite precipitates after immersion in phosphate-buffered saline., Conclusions: The experimental material possessed adequate physicochemical properties, low cytotoxicity and good calcium phosphate nucleation., (© 2017 International Endodontic Journal. Published by John Wiley & Sons Ltd.)

Many studies have been carried out to examine whether there is an association between CDKN2A polymorphisms and cancer risk, but the results from these studies were controversial. In the present study, we performed a meta-analysis to clarify whether there is an association of CDKN2A polymorphisms and cancer risk. Published reports were searched in PubMed and Google Scholar. ORs with 95% CIs were calculated in the dominant models. Twenty six case-control studies that met the inclusion criteria were included in the final meta-analysis. Overall, we found that rs3731249, rs11515, and rs3088440 polymorphisms were not associated with cancer risk (OR=1.27, 95%CI: 0.79-2.03; OR=0.91, 95%CI: 0.79-1.03; OR=1.02, 95%CI: 0.95-1.09). However, CDKN2A rs3731249 polymorphism was significantly associated with ovarian cancer risk (OR=0.78, 95%CI: 0.65-0.95). A significant association was observed in Asian with rs11515 polymorphism (OR=0.48, 95%CI: 0.28-0.83). This meta-analysis shows that CDKN2A rs3731249 polymorphism was significantly associated with ovarian cancer risk. In addition, CDKN2A rs11515 polymorphism might associate with cancer risk in Asians.

Kirchhoff thin elastic rod models are important in the study of the mechanism determining the configurations of flexible structures not only at the macroscopic but also at the microscopic scale. In this study, the energy balance method has been well applied to analyze the configuration of a DNA elastic rod in the presence of interfacial traction. An approximate solution for the shape equations has been obtained, and the relationship between the interfacial factor and the configuration of the DNA segment is derived. The results may provide an explanation for the onset of the formation of kinks in DNA when immersed in a solution.

In this study, various amounts of benzophenone-3 (BP-3), a solid-type of organic UV-filter, were encapsulated in mesoporous silica (MS) to form the BP-3 encapsulated by MS UV-filters (BESs), BES-1 and BES-2, via in-situ sol-gel process. The characterization of BESs was completed using Fourier transform infrared (FTIR) spectroscopy, thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC). The results showed that of the BES filters, BES-2 containing emulsion (BES-2-E) exhibited about 2 times and 1.64 times higher SPF and erythemal UV-A PF values, respectively, and after 3 months about 7-8 times higher protection retention capability than the free BP-3 containing emulsion (BP-3-E). Moreover, the result of the in vitro skin penetration test using Franz glass diffusion cell indicated that the skin permeation of BP-3 from BESs was about 3 times lower than from BP-3-E. This property is particularly important for sunscreens because the amount of sunscreen penetration inside the stratum corneum directly correlates to its UV protection ability, and consequently its ability to reduce phototoxic and photo-allergic reactions that are damaging to the skin. The results of this study demonstrated the potential of as-prepared BES-2 as a UV-filter for cosmetic products., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Objective: The use of dye substances in hair dye products has led to controversial public and scientific discussions about their impact on human health. This study aimed to explore a rapid method for identification of dye substances in hair dye products., Methods: Thin layer chromatography (TLC) method was conducted in this study. We developed basic data of 32 dye substances by 4 developing solvents and 2 indicator sprays. The dye substances were identified by comparing the Rf values and colors between samples and reference substances., Results: Thirty samples were identified by the established method. 22 hair dye substances were detected in 16 formula known samples, 15 hair dye substances were detected in 14 formula unknown samples. Four laboratories participated in the validation and the results showed good interlaboratory reproducibility., Conclusion: The developed TLC method is simple, rapid, reliable and can be finely used for identification of dye substances in hair dye products., (© 2014 Society of Cosmetic Scientists and the Société Française de Cosmétologie.)

Background: This study was designed to assess the neuroprotective effect of xenon-induced delayed postconditioning on spinal cord ischaemia-reperfusion injury (IRI) and to determine the time of administration for best neuroprotection in a rat model of spinal cord IRI., Methods: Fifty male rats were randomly divided equally into a sham group, control group, and three xenon postconditioning groups (n=10 per group). The control group underwent spinal cord IRI and immediately inhaled 50% nitrogen/50% oxygen for 3 h at the initiation of reperfusion. The three xenon postconditioning groups underwent the same surgical procedure and immediately inhaled 50% xenon/50% oxygen for 3 h at the initiation of reperfusion or 1 and 2 h after reperfusion. The sham operation group underwent the same surgical procedure without aortic occlusion, and inhaled 50% nitrogen/50% oxygen. Neurological function was assessed using the Basso, Beattie, and Bresnahan score at 4, 24, and 48 h of reperfusion. Histological examination was performed using Nissl staining and immunohistochemistry, and apoptosis was detected by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labelling staining., Results: Compared with the control group, the three xenon postconditioning groups showed improvements in neurological outcomes, and had more morphologically normal neurones at 48 h of reperfusion. Apoptotic cell death was reduced and the ratio of Bcl-2/Bax immunoreactivity increased in xenon-treated rats compared with controls., Conclusions: Xenon postconditioning up to 2 h after reperfusion provided protection against spinal cord IRI in rats, but the greatest neuroprotection occurred with administration of xenon for 1 h at reperfusion., (© The Author [2013]. Published by Oxford University Press on behalf of the British Journal of Anaesthesia. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

In the present study, about 45 and 34 wt% of benzophenone-3 (BP-3), an organic UV filter, was adsorbed on a high surface area mesoporous silica (MS) drug carrier to prepare BP-3-bearing MS (MSBP) sunscreen materials MSBP-1 and MSBP-2, respectively. The effect of the adsorption of BP-3 by MS on the UV protection ability of MSBP was demonstrated and a synergistic UV protection effect was observed in the as-prepared MSBP UV filters. Compared with free BP-3, adsorbed BP-3 had greatly reduced crystallinity and the dispersion of MSBP was significantly improved in the sunscreen. The in vitro sun protection factor (SPF) and in vitro UV-A values of the MSBP-2-based sunscreen was about 17.3% and 17.0% higher than that of free BP-3-based sunscreen, respectively, indicating that the ability of the sunscreen to protect against UV-B and UV-A improved because of the BP-3 content of the MS matrix. In addition, the decrease in SPF and UV-A values over time was significantly less in the MSBP-based sunscreens than in free BP-3-based sunscreen. Results of this study reveal that MS is a promising organic sunscreen carrier as well as a potential carrier for other topical drugs., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Background: The neuroprotective effects of xenon post-conditioning following spinal cord injury remain unknown. We monitored the effect of xenon post-conditioning on the spinal cord following ischaemia-reperfusion injury and determined its mechanism of action., Methods: Spinal cord ischaemia was induced following balloon occlusion of the thoracic aorta in male Sprague-Dawley rats. Rats were divided into three groups (n = 30 in each group). The control group underwent ischaemia-reperfusion injury and immediately inhaled 50% (v/v) nitrogen at the time of reperfusion for 60 min continuously. The xenon-post-conditioning group underwent the same surgical procedure and immediately inhaled 50% (v/v) xenon at the time of reperfusion for 60 min continuously. The sham operation group underwent the same surgical procedure without aortic catheter occlusion and inhaled the same gas as that in control rats. Neurologic function was assessed using the Basso, Beattie, and Bresnahan score at 4, 24, and 48 h after reperfusion. Histological changes were observed using Nissl staining, the ultrastructure of the spinal cord was examined using transmission electron microscopy, and apoptosis was monitored using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelling., Results: Compared with the control group, the xenon-post-conditioning group showed improved neurologic outcomes (11.3 ± 1.6 vs. 15.7 ± 3.1, respectively) and had more morphologically normal neurons (6 ± 2 vs. 12 ± 3) at 48 h after reperfusion. Moreover, apoptotic cell death in xenon-treated rats was reduced when compared with control rats (18.29 ± 3.06 vs. 27.34 ± 3.63, P < 0.05, respectively)., Conclusions: Xenon post-conditioning exerts a neuroprotective effect on the spinal cord following ischaemia-reperfusion injury via its anti-apoptotic role., (© 2012 The Authors. Acta Anaesthesiologica Scandinavica © 2012 The Acta Anaesthesiologica Scandinavica Foundation.)

A simple and direct reversed-phase high performance liquid chromatography (RP-HPLC) method with UV detection was developed and validated for the determination of mono- and di-D-α-tocopherol polyethylene glycol 1000 succinate (TPGS 1000) in TPGS mixture. Before the HPLC analysis, mono- and di-TPGS 1000 were separated by simulated moving bed (SMB) chromatography system and characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The mass spectrometric results confirmed that the molar mass distribution of TPGS prepared in our laboratory was very close to that of the product of Eastman Chemical Company with similar n¯ (average polymerization degree), M(n)¯ (number-average molecular weight) and M(w)¯ (weight-average molecular weight). The HPLC analysis was carried out on a C30 analytical column with mobile phases comprised of acetonitrile (A) and isopropanol (B) in gradient conditions. Validation of the analytical method was done on the following parameters: system suitability, linearity, limits of detection and quantification, accuracy and precision, method robustness and solution stability. The linearity of the calibration curves for mono- and di-TPGS 1000 from both sources was found to be good (r(2)>0.9996). The recovery values were from 94.6% to 103.3% for mono-TPGS, and 93.5% to 103.3% for di-TPGS. This method could be successfully used in the direct quantification of mono- and di-TPGS in TPGS 1000 mixture using TPGS standards with similar molecular mass distributions although derived from different sources., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Background: Most publications to date on comorbidities associated with psoriasis have focused on cardiovascular and metabolic diseases. Few comprehensive investigations of medical comorbidities in a cohort of patients with psoriasis appear in the literature., Objectives: To examine the prevalence of comorbidities in adult patients with psoriasis, including a comparison of comorbid prevalence vs. that in controls without psoriasis, in a nationally representative dataset in Taiwan., Methods: There were 1685 adult patients with psoriasis in the study group and 5055 randomly selected subjects in the comparison group. We used conditional logistic regression analyses to examine the risk of 29 comorbidities for these two groups after adjusting for monthly income, geographical region of residence and the level of urbanization of each patient's community of residence., Results: After adjusting for several potential confounders, patients with psoriasis had higher odds of comorbid congestive heart failure [odds ratio (OR) 1·63], ischaemic heart disease (OR 1·51), renal failure (OR 1·45), uncomplicated diabetes (OR 1·37), liver diseases (OR 1·34), hepatitis B or C (OR 1·34), complicated diabetes (OR 1·32), hyperlipidaemia (OR 1·28), hypertension (OR 1·24) and peptic ulcer (OR 1·22) than did patients without psoriasis. However, patients with mild psoriasis had higher odds of comorbidity only with uncomplicated diabetes (OR 1·55), asthma (OR 1·30), liver diseases (OR 1·30) and peptic ulcer (OR 1·26) than patients without psoriasis., Conclusions: We conclude that psoriasis is associated with a variety of medical comorbidities including cardiovascular diseases, metabolic diseases, renal failure, liver diseases, viral hepatitis B or C, asthma and peptic ulcers., (© 2011 The Authors. BJD © 2011 British Association of Dermatologists.)

Background: Eczema has been shown to be associated with an increased risk of depression. However, the effect of cigarette smoking on the association between eczema and depression remains unclear., Objectives: In this study, we investigated whether smoking behaviour and tobacco exposure influence the association between eczema and depression., Methods: This was a population-based cross-sectional study. Subjects (287 eczema patients and 40 patients with depression, out of a total of 2974 subjects in the database) were from the National Health and Nutrition Examination Survey 2005-2006, aged between 20 and 59 years. Lifetime presence of eczema was obtained by self-reporting questionnaires, and depression was assessed by the Patient Health Questionnaire. Smoking status was determined by self-report and serum cotinine levels. Univariate and multivariate logistic regressions were used to assess the association between eczema and depression with and without adjusting for smoking behaviour. Stratified analysis was also performed according to smoking status., Results: Eczema was significantly associated with depression (adjusted OR: 2.12; 95% CI: 1.59-2.83). This association persisted after additionally adjusting for smoking status. In stratified analysis, the association between eczema and depression was higher and stronger among current smokers than never smokers, former smokers and passive smokers., Conclusion: Our findings indicated that cigarette smoking may modify the risk of depression in eczema. We suggested cessation of smoking in eczema patients to decrease the risk of this psychiatric co-morbidity., (© 2010 The Authors. Journal of the European Academy of Dermatology and Venereology © 2010 European Academy of Dermatology and Venereology.)

Background: Psychiatric disorders have been shown to be associated with impaired immune response, including decreased cellular immunity to varicella-zoster virus. However, the risk of herpes zoster (HZ) in psychiatric patients is, to date, unknown., Objective: The aim of this study was to determine the risk of herpes zoster (HZ) in psychiatric patients compared with the general population., Methods: We used data from the Taiwan Longitudinal Health Insurance Database from 2004 to 2006. Our study cohort consisted of patients aged 18 years and older diagnosed with psychiatric disorders in 2004 (N=42 340). The comparison cohort (N=169 360) consisted of four age- and gender-matched controls randomly selected for every patient in the study cohort. All subjects were followed from the date of cohort entry until they developed HZ or the end of 2006, whichever was earliest. Stratified Cox proportional hazard regressions were performed to compute the 2-year HZ-free survival rates., Results: After adjusting for potential confounders, we found patients with psychiatric disorders were more likely to have an episode of HZ than the control population [adjusted hazard ratio (HR), 1.29; 95% confidence interval (CI), 1.18-1.38]. When stratified by age and psychiatric diagnostic categories, in patients aged ≤60 years, the adjusted HRs for HZ were 1.34 (P=0.026) for patients with affective psychoses, 1.42 (P<0.001) for those with neurotic illness or personality disorders and 1.53 (P<0.001) for patients with other mental disorders. However, in patients aged >60years, only neurotic illness or personality disorders were significantly associated with an increased risk of HZ (adjusted HR, 1.26; P=0.003)., Conclusions: Our analysis suggests that patients with psychiatric disorders are at increased risk of HZ, especially those aged ≤60 years. Further study is required to elucidate the nature of this association., (© 2010 The Authors. Journal of the European Academy of Dermatology and Venereology © 2010 European Academy of Dermatology and Venereology.)

Background: Breastfeeding is undisputedly preferable to formula feeding for infant nutrition because of its nutritional, immunological and psychological benefits. However, studies on the association between breastfeeding and development of atopic dermatitis (AD) have shown inconsistent results., Objectives: To examine the association between exclusive breastfeeding for at least 3 months after birth and the development of AD in childhood., Methods: An electronic literature search of MEDLINE (January 1966-May 2008) and EMBASE (1980-May 2008) was conducted. Prospective cohort studies that met the predetermined criteria were independently assessed by three reviewers. The pooled effect estimate was calculated by random effects model. Heterogeneity across the studies was investigated by meta-regression analysis., Results: Twenty-one studies with 27 study populations were included for meta-analysis. The summary odds ratio (OR) for the effect of exclusive breastfeeding on the risk of AD was 0.89 (95% confidence interval, CI 0.76-1.04). Heterogeneity was found across the studies (chi(2) = 83.6, d.f. = 26; P < 0.001). Breastfeeding was associated with a decreased risk of AD (OR 0.70; 95% CI 0.50-0.99) when analysis was restricted to the studies comparing breastfeeding with conventional formula feeding. The pooled OR for study populations with atopic heredity was 0.78 (95% CI 0.58-1.05)., Conclusions: There is no strong evidence of a protective effect of exclusive breastfeeding for at least 3 months against AD, even among children with a positive family history.

Objective: To identify the aetiologies of pulmonary cavities and the clinical predictors of cavities of mycobacterial origin., Setting: A tertiary referral hospital in South Korea, where the prevalence of tuberculosis (TB) is intermediate., Design: A retrospective review of clinical records and radiographic examinations of patients presenting pulmonary cavities on simple chest radiograph between January and December 2005., Results: Of 131 patients enrolled with pulmonary cavities, 66 (50.4%) had cavities of mycobacterial origin. Age <50 years (P = 0.04) and largest cavity located in the upper lobes (P = 0.04) increased the likelihood that the cavities were of mycobacterial origin. Conversely, history of malignancy (P = 0.02), lesions confined to one lobe (P = 0.02) and multiple enlarged mediastinal lymph nodes (P = 0.03) suggested a non-mycobacterial cause., Conclusion: Mycobacterial infection accounted for half of the cavitary lesions identified in this study. In older patients with a history of malignancy, non-nodular infiltration, lesions confined to one lobe and with multiple lymphadenopathy, diseases not caused by mycobacteria should be considered.

Soluble or sub-unit protein vaccines alone are incapable of generating antigen-specific cellular immune responses. This failure can be attributed to the manner in which the immune system processes antigen; endogenous antigens are cycled through the MHC class I pathway to stimulate CD8+ restricted responses and exogenous antigens are processed through the MHC class II pathway to generate humoral immunity. Traditionally sub-unit vaccines have been formulated with adjuvants to enhance immunogenicity, however in the last decade a number of adjuvants have been developed that effectively stimulate the generation of both humoral and cellular immune responses, although the manner in which they exert their effects has not been investigated. Here we describe Tomatine, a glycoalkaloid based adjuvant, capable of stimulating potent antigen-specific humoral and cellular immune responses that contribute to protection against malaria, Francisella tularensis and regression of experimental tumors. Using in vivo models we investigated the manner in which cellular immune responses were generated by Tomatine. We established that Tomatine did not require either lymph node or splenic macrophages to generate cytotoxic T lymphocytes (CTL) and delivered soluble protein into a pathway not dependant on the machinery of the classical MHC class I pathway. We also observed that at the molecular level Tomatine required both CD80 and CD86 costimulation to engender antigen-specific cellular immunity.

Background: A double-blind, randomized study was designed to compare the recovery manner of mivacurium infusion with or without edrophonium reversal in microscopic laryngeal surgery. Neuromuscular blockade was quantified using the train-of-four stimuli to the ulnar nerve and quantification of the ratio of the fourth twitch to the first twitch., Methods: With the approval of the Human Studies Committee of the Taipei Veterans General Hospital and patient informed consent, 40 healthy (ASA I or II) patients with age from 24 to 54 years, undergoing microscopic laryngeal surgery were randomly selected for study. Mivacurium chloride 0.2 mg/kg was given intravenously, and then it was given in continuous infusion to maintain muscle relaxation at 90% twitch block during the procedure. At the end of operation, mivacurium infusion was terminated. In a double-blind manner, group I patients (n = 20) received intravenous edrophonium 1 mg/kg and atropine 0.01 mg/kg for reversal when T1 was at 10% recovery whereas patients in Group II (n = 20) received placebo in the same manner. Mean infusion rate, recovery index (RI50, time from T1 25% to T1 50%; RI75, time from T1 25% to T1 75%), extubation time, and discharge time between groups were compared. Nausea, vomiting, and dysrhythmias were also documented until the patient was discharged from hospital., Results: The demographic data between two groups were similar. The recovery index (RI75) for group I was shorter than that of placebo group (5.3 +/- 2.19 min vs. 7.3 +/- 0.9 min) and the difference was statistically significant (P = 0.017). There were no statistically significant differences in mean infusion rate, incidence of nausea and vomiting, and discharge time from the POR. The incidence of tachycardia or arrhythmia in group I was significantly greater than that in group placebo., Conclusions: Mivacurium, a short-acting nondepolarizing agent, is a suitable muscle relaxant for patients receiving microscopic laryngeal surgery. Recovery time with the use of edrophonium as reversal agent was shorter than with placebo, but extubation and discharge time did not differ in two groups. The time which could be saved by the use of edrophonium for reversal of mivacurium to hasten the maximal recovery appears to be less than a few minutes. Therefore, clinically, the value of routine use of edrophonium to obtain a faster recovery does not outweigh its demerits of cost and risk and is not worthy of recommendation.

Experimentally, CCl4 was effectively mineralized by CuO to yield stable inorganic species of CO2 and CuCl2 (CCl4 + 2CuO --> 2CuCl2 + CO2). High CCl4 conversions (63-83%) were found in the mineralization process performed at 513-603 K for 10-30 min. Using X-ray-absorption near edge structure (XANES) and X-ray photoelectron spectroscopies, we found that most CuCl2 was encapsulated in the CCl4-mineralized product solid (mineralization at 513 K for 30 min). At higher mineralization temperatures (563-603 K), CuCl2 was found to be predominant on the surfaces of the mineralization product. Speciation of copper in the mineralization product solid was also studied by extended X-ray absorption fine structure (EXAFS) spectroscopy. Bond distances of Cu-O and Cu-Cl in the CCl4-mineralized product solid were 1.93-1.94 and 2.10-2.12 , respectively, which were greater than those of normal CuO and CuCl2 by 0.03-0.07 A. The increase of the bond distances for Cu-O and Cu-Cl might be due to Cl insertion and concomitant structural perturbation of unreacted CuO in the mineralization process. Forthe second shell around copper atom, bond distances of Cu-(O)-Cu also increased by 0.03-0.05 A, and the coordination numbers of Cu-O and Cu-(O)-Cu decreased, as expected, in the mineralization process. In addition, stoichiometrically excess oxygen atoms were found on the solid surfaces, and they might play an important role in the mineralization of CCl4, leading to the formation of CO2 and Cl. Chloride atoms might be further captured by CuO, yielding CuCl2 in the mineralization process. This work exemplifies the utilization of X-ray spectroscopies (XANES, EXAFS, and XPS) to reveal the speciation and possible reaction pathway in a very complex mineralization process in detail.

Purpose: The purpose of this study was to evaluate glucose responsiveness in HepG2 human hepatoma cells transduced by a recombinant adeno-associated virus (rAAV) vector containing the insulin gene promoter. and to investigate the effect of protamine sulfate on rAAV-mediated gene delivery., Methods: Recombinant AAV vector, AAV.Ins.Luc.delta EGFP, was employed to transduce HepG2 hepatoma cells. Virus infection was carried out either in the absence or presence of protamine sulfate, followed by fluorescence microscopic examination, luciferase activity assay, and flow cytometric analysis. Electrokinetic measurements were carried out to determine the effect of protamine sulfate on zeta potential of the cells and the virus., Results: Glucose-responsive luciferase gene expression was obtained in rAAV-transduced HepG2 cells. Addition of 5 microg/ml protamine reversed the zeta potential of the cells and the virus particles, leading to enhanced transgene expression in rAAV-transduced HepG2 cells. Enhancement of protamine sulfate on rAAV-mediated gene transfer was dose-dependent. Addition of more than 5 microg/ml protamine resulted in a reduction of infectability of the virus., Conclusions: Glucose responsiveness in the millimolar concentration range can be obtained in rAAV-transduced HepG2 cells. Protamine sulfate, up to 5 microg/ml, enhanced the rAAV transduction efficiency in HepG2 cells. The enhancement was correlated with zeta potential of the cells and the virus.

Speciation of copper in the fly ash solidification process has been studied by X-ray based spectroscopies inthe present work. Fourier transformed EXAFS (extended X-ray absorption fine structural) spectra of the solidified fly ashes showed that the bond distance of Cu-O (first shell) was 1.96 A with a coordination number (CN) of about 3.0. However, in the second shell of copper atoms, the bond distance of Cu-(O)-Cu was decreased by 0.12-0.22 A during solidification, which might cause the stabilization of the CuO species in the solidified fly ash matrix. By the least-squares fits of the XANES (X-ray absorption near edge structural) spectra, fractions of the main copper species in the solidified fly ashes such as CuCl2 (0.08-0.11), Cu2O (0.07-0.09), Cu(OH)2 (0.31-0.33), and CuO (0.49-0.52) were observed. Combined EXAFS and XANES observations suggested that chemical reactions such as hydroxylation of CuCl2 and oxidation of Cu2O and/or metallic Cu might involve in the solidification process, which also led to a significant reduction of the leachability of copper from the solidified fly ashes.

To employ hepatocytes as surrogate beta-cells for gene therapy of diabetes, a regulatory system was devised in this study by placing the human insulin cDNA under the control of the phosphoenolpyruvate carboxykinase (PEPCK) promoter, followed by the cytomegalovirus immediate early promoter-driven enhanced-green-fluorescent-protein open reading frame. The expression cassette was inserted into the adeno-associated virus vector between two inverted terminal repeats, and used to produce recombinant adeno-associated virus (rAAV). HepG2 human hepatoma cells were transduced by rAAV at the desired multiplicity of infection, followed by treatment with various concentrations of retinoic acid, dexamethasone, dibutyryl cAMP (dbcAMP) and 3-isobutyl-1-methylxanthine (IBMX). The cell-culture media were collected at 8, 16 and 24 h later. Proinsulin/insulin levels were determined with human proinsulin/insulin radioimmunoassay kits. Transduction of HepG2 cells by rAAV showed that green fluorescence was produced as early as 12 h after rAAV infection. Flow-cytometrical analysis demonstrated that transduction efficiency increased with the numbers of transducing rAAV particles used. The transduced hepatocytes were shown to secrete immunoreactive proinsulin/insulin, which were stimulated by the concentrations of retinoic acid, dexamethasone and dbcAMP in the culture medium. High conversion from proinsulin into insulin occurred when these cells were treated with dexamethasone and dbcAMP. The presence of IBMX enhanced the secretion of proinsulin/insulin from the dbcAMP-treated cells. We conclude that rAAV is a promising vector for gene therapy of diabetes. Regulated secretion of proinsulin/insulin can be obtained in the rAAV-transduced HepG2 cells conferred with the PEPCK promoter via rAAV-mediated gene transfer.

XANES fitting indicates that non-Cr2O3 form of Cr(III), transformed from CrO3 by four-hour heat application at 1100 degrees C, is the key species sorbed onto or lattice-diffused into kaolin matrices that exhibits strong leaching resistance to sequential extraction using various combined solutions including HNO3, H2O2, and HF. The EXAFS spectra are all in agreement with the XANES results. It is suggested that some Cr2O3 might have reacted with kaolin to form new compounds such as Cr2O3 x Al2O3 x 2SiO2 to account for the slight dissimilarity between the EXAFS spectra of samples and that of Cr2O3.

Thermal immobilization of copper contaminants in soil analogue minerals, quartz and kaolin, at low temperatures such as 300 degrees C is studied to corroborate its technical feasibility as a method for soil remediation. We use a synchrotron-based, X-ray absorption spectroscopy (XAS) technique to study the speciation of and the local structure around copper in the soil analogues that are thermally treated at 300-900 degrees C for 1 h. The toxicity characteristic leaching procedure (TCLP) method is employed to investigate the leaching behavior of copper compounds. CuO, being predominately transformed from Cu(OH)2 with a lesser amount from Cu(NO3)2 by 1-h heat application at 300-900 degrees C, is identified by the spectroscopy of X-ray absorption near-edge structure (XANES) and extended X-ray absorption fine structure (EXAFS) as the key species that is leaching-resistant due to its low solubility and its chemisorption onto the soil analogue minerals. Fourier transform of EXAFS spectrum of the Cu-doped kaolin heated at 900 degrees C for 1 h indicates that the intensity of Cu-Cu peaks (2.50 and 5.48 A, both without phase shift correction) is either relatively smaller or disappearing as compared with that of kaolin samples heated at 300 and 500 degrees C. The EXAFS analysis suggests that the Cu solid phase in the 900 degrees C kaolin sample is different from the lower temperature samples, the 900 degrees C SiO2 sample, and the Cu standards. The leaching studies also support the formation of a less soluble phase in the 900 degrees C kaolin sample. An increase of heating temperature, in the range of 105-900 degrees C, reduces the Cu leaching percentage; this reduction trend is more marked for Cu-doped kaolin than for Cu-doped SiO2.

Purpose: Recent progress in genetic engineering presents the possibility of providing physiologically regulated glucose metabolism in individuals with diabetes. The objective of this study is to explore the feasibility of obtaining glucose dependent gene expression in the pancreatic beta-cell lines via recombinant adeno-associated virus type 2 (rAAV) mediated gene transfer., Methods: Two transcription cassettes containing the luciferase gene under the control of the rat insulin I gene promoter and the enhanced green fluorescent protein (EGFP) open reading frame under the control of the immediate early gene promoter of human cytomegalovirus (CMV) were placed in series between the inverted terminal repeats (ITRs) of AAV. The rAAV vectors produced were used to transduce pancreatic beta-cell line grown in the absence or presence of various concentrations of glucose. Luciferase activity assays were performed at 72 hr post-transduction., Results: Glucose-responsive reporter gene expression was obtained in both calcium phosphate transfected HIT-T15 and betaHC-9 cells, demonstrating regulated luciferase gene expression under control of the insulin gene promoter. At MOI of 100, rAAV-transduced betaHC-9 cells exhibited glucose-dependent luciferase activities, which were approximately 4.3 fold higher than those transfected by the calcium phosphate coprecipitation method at 20 mM glucose., Conclusions: Delivery of the insulin gene promoter via rAAV was shown in this study to result in glucose-dependent control of the reporter gene expression. The results suggest that rAAV is an efficient viral vector for gene transfer into the pancreatic islet cells.

In this study we attempted to investigate the feasibility of transferring exogenous DNA into the recipient host via polyomavirus major capsid protein VP(1) pseudocapsids generated from recombinant Escherichia coli, designated PyVP(1,E), both in vitro and in vivo. NIH-3T3 and FR3T3 cells were transfected with pCMV beta and pPyMT-1 plasmid DNA, respectively. In vitro DNA transfection was carried out via Pseudofect- and PyVP(1,E)-mediated methods, or by co-precipitation with calcium phosphate. Expression of beta-galactosidase and PyMT reporter genes was examined by Western-blot analysis. Parallel experiments were performed in vivo by direct injection of pCMV beta and pPyMT-1 plasmid DNA, complexed with PyVP(1,E), into the livers of Wistar rats, followed by Western-blot analysis and histochemical staining. The results obtained from in vitro transfection experiments showed that expression of the reporter genes can be detected in the recipient cells at 48 h post-transfection. PyVP(1,E) was shown to exhibit similarly efficient in vitro DNA transfection properties to Pseudofect, which was obtained from recombinant baculovirus.

Behavior of Br in pyrolysis of the printed circuit board waste with valuable copper and oil recycling has been studied in the present work. Experimentally, pyrolysis of the printed circuit board waste generated approximately 40.6% of oils, 24.9% of noncondensible gases and 34.5% of solid residues that enriched in copper (90-95%). The cuts of the oils produced from pyrolysis of the printed circuit board waste into weighted boiling fraction were primarily light naphtha and heavy gas oil. Approximately 72.3% of total Br in the printed circuit board waste were found in product gas mainly as HBr and bromobenzene. However, by extended X-ray absorption fine structural (EXAFS) spectroscopy, Cu-O and Cu-(O)-Cu species with bond distance of 1.87 and 2.95 A, respectively, were observed in the solid residues. Essentially, no Cu-Br species was found.

The phylogenetic relationships of nine genera in four tribes of the family Brassicaceae were estimated from the sequences of the internal transcribed spacer region (ITS) of the 18S-25S nuclear ribosomal DNA. The entire ITS region of 16 accessions belonging to 10 species of seven genera was sequenced. Eight published sequences of Brassicaceae were also used. A total of 27 sequences were included in this study; four of them were found to be pseudogenes. Both the neighbor-joining and the parsimony trees suggest that the nine genera can be divided into three groups: (1) Arabidopsis, Cardaminopsis, Capsella, and Lepidium; (2) Rorippa and Cardamine; and (3) Brassica, Sinapis, and Raphanus. In contradiction to the proposal that Cardaminopsis and Arabidopsis be put into an expanded tribe Arabideae, our data show that these two genera are more closely related to Capsella and Lepidium (tribe Lepidieae) than to Rorippa and Cardamine (tribe Arabideae). Further, our data show that within the tribe Brassiceae, Raphanus is more closely related to B. nigra than to the B. oleracea/B. rapa clade. This result is in agreement with the nuclear data obtained in several studies, but is in conflict with the RFLP data of mitochondrial and chloroplast DNA. As pointed out by previous authors, it is possible that Raphanus is a hybrid between the B. nigra and B. oleracea/B. rapa lineages with the latter as the maternal parent., (Copyright 1999 Academic Press.)

We obtained 16 nucleotide sequences ( approximately 1400 bp each) of the first intron of the mitochondrial (mt) gene for NADH subunit 4 (nad4) from 10 species of Brassicaceae. Using these new sequences and five published sequences from GenBank, we constructed a phylogenetic tree of the Brassicaceae species under study and showed that the rate of nucleotide substitution in the first intron of nad4 is very low, about 0.16-0.23 x 10(-9) substitution per site per year, which is about half of the silent rate in exons of nad4. The ratios of substitution rates in this intron, ITS, and IGS are approximately 1:23:73, where ITS is the nuclear intergenic spacer between 18S and 25S rRNA genes and IGS is the intergenic spacer of 5S rRNA genes. A segment (335 bp) in the first intron of nad4 in Brassicaceae species that is absent in wheat was considered as a nonfunctional sequence and used to estimate the neutral rate (the rate of mutation) in mtDNA to be 0.5-0.7 x 10(-9) substitution per site per year, which is about three times higher than the substitution rate in the rest of the first intron of nad4. We estimated that the dates of divergence are 170-235 million years (Myr) for the monocot-dicot split, 112-156 Myr for the Brassicaceae-Lettuce split, 14.5-20.4 Myr for the Brassica-Arabidopsis split, and 14.5-20.4 Myr for the Arabidopsis-Arabideae split.

Conformations of polyomavirus (Py) major capsid protein VP1 were analyzed by circular dichroism (CD) and fluorescence spectroscopy in the presence of sodium dodecyl sulfate (SDS). Binding of PyVP1 to SDS induced marked conformational changes of PyVP1, which were reflected on the CD and fluorescence spectra. Abrupt changes in both optical properties occurred within the narrow ranges of SDS concentrations with the transition midpoints closely related to SDS micelle formation. Analysis of circular dichroism spectra showed that the contents of alpha-helices, beta-sheets, beta-turns and random coils in PyVP1 varied upon addition of SDS, demonstrating the exquisite sensitivity of the conformations of the protein to the environment. The interactions of PyVP1 with SDS were shown to be dependent on the ionic strength of the protein solution, suggesting that both hydrophobic and electrostatic forces contribute to the PyVP1-SDS complex formation. The SDS-induced conformational changes of PyVP1 appeared to be a two-stage process.

The conformational changes of polymavirus (Py) major capsid protein VP1 in solution by the solution pH, addition of calcium, and ionic strength were examined by circular dichroism (CD) and fluorescence spectroscopy. Comparison of the predicted secondary structures of PyVP1 and simian virus (SV) 40 by the methods of Chou-Fasman, Garnier et al., and Yang method are presented. Hydropathicity, surface probability, and chain flexibility of PyVP1 were computer-analyzed by the methods of Kyte and Doolittle, Emini et al., and Karplus and Schulz, respectively. The CD measurements indicate that the secondary structure of PyVP1 is little dependent on its concentration, Ca2+ concentration, and ionic strength, but is strongly pH dependent. Fluorescence studies showed that emission spectra of PyVP1 are also pH-dependent. At extreme acidic and alkaline pH, the fluorescence intensity of PyVP1 is decreased and the emission maximum is red-shifted. The fluorescence of PyVP1 is quenched by the presence of CsCl, KI, and acrylamide. The analyses of the modified Stern-Volmer plots indicate that five of seven tryptophan residues in PyVP1 are located on the surface of the protein, among which two are accessible to Cs+ and the other three are accessible to I- . The two others are buried more deeply in the interior of the protein molecule.

Based on the electrical properties of the skin, a method employing the unijunction transistor (UJT) relaxation oscillator for detecting low skin resistance points (LSRP) was developed in this study. By means of this instrumentation, the topography of the LSRP in Wistar rats was developed. All the LSRP in the rats were found to be bilaterally and symmetrically distributed except those points located on the dorsal midline (i.e., governor vessel, GV) and the ventral midline (i.e., conception vessel, CV). The resistances of the LSRP on these two major vessels, including 14 CV points and 17 GV points of six rats were experimentally determined to be in the ranges of 179.4 +/- 41.2 K omega and 152.5 +/- 32.2 K omega, respectively. The resistances of the GV points were found in general to be lower than those of the CV points. Most non-LSRP, on the other hand, exhibited resistances of greater than 420 K omega. It is noted that the resistances of most LSRP increased yet still retained a separate identity within thirty minutes after the death of the animals, but the low resistance properties of some LSRP gradually disappeared thereafter and could not be detected by the relaxation oscillator.

Human gastric cancer MKN-45 cells were transfected with pULB 3238, a plasmid carrying MVMp NS-1 gene with its original P4 promoter replaced by the glucocorticoid inducible promoter MMTV-LTR. After the integration and expression of NS-1 gene, some of the transfectants died, while others remained alive, but the growth features of survived cells were changed. For further study on the antineoplastic function of parvoviral NS-1 protein in vivo, transgenic mice carrying NS-1 genes were established by conventional method. Among 4 founders, one of them was found to be able to transmit the transgene to around 50% of their offsprings. RT-PCR was performed to indicate the expression of NS-1 gene in transgenic mice and its mRNA appeared in a variety of tissues. The expression of integrated NS-1 gene may correlate with the decreased incidence of tumor induced in vivo by chemical carcinogens.

The middle T antigen (MTAg) encoded by polyomavirus plays an important role in virus-mediated tumorigenesis. The activated protein kinase activity of MTAg-associated pp60c-src has been shown to be necessary for cell transformation by polyomavirus. In this study, the effects of herbimycin A on the pp60c-src kinase activities in the polyomavirus- and MTAg-transformed cells were studied. Phosphorylation of src and MTAg is reduced in polyomavirus and MTAg- transformed cells pretreated with herbimycin A. Inactivation of the enzymatic activity by herbimycin A was found to be dependent on the time of incubation and the drug concentration. In contrast, src immunoprecipitates from untreated MTAg-transformed cells appeared to be resistant to inhibition by herbimycin A. Herbimycin A does not affect the synthesis of MTAg and pp60c-src in the MTAg-transformed cells. These results suggest that pp60c-src kinase activity in the drug-treated cell lysates is more sensitive to herbimycin A inhibition than the same activity in the immunoprecipitates.

DEAE-dextran-mediated gene transfer was studied for the introduction of pSV2neo DNA into Fisher-rat 3T3 (FR3T3) cells. Zeta (zeta) potentials of the DEAE-dextran-DNA complexes and FR3T3 cells were found to be dependent on the concentration of DEAE-dextran in the medium. The maximum transfection efficiency occurred at a DEAE-dextran/DNA ratio of 50:1 or thereabouts. The interaction between DNA and cells is determined by the adsorption process. The results obtained, along with the correlation between the kinetic adsorption behaviour of 3H-labelled DNA and the transfection efficiency, indicated that adsorption of DEAE-dextran-DNA complexes to the negatively charged cell surfaces, due to electrostatic and dispersion attraction, plays the decisive role in determining the DNA transfection efficiencies.

Electropulsation has been proved to be a powerful technique for DNA transfection in vitro. This study was undertaken to assess the feasibility and evaluate the potential of applying this approach for facilitating gene transfer in vivo. The pCMVβ plasmid, containing the Escherichia coli lacZ open reading frame under the cytomegalovirus early promoter, was complexed with poly-L-lysine (PLL) and injected directly into the livers of Wistar rats; electropulsation was then applied either at the low skin resistance points (LSRPs) of the skin, the non-LSRP, or directly at the liver by either a portable electrostimulator or an electropulsation unit. The locations of the LSRPs were detected by the unijunction transistor (UJT) relaxation oscillator. Seventy-two hours after gene transfer, the animals were sacrificed and the livers were excised, followed by examination of the transgene expression. The results obtained from X-gal staining, in situ hybridization, and Western blot analysis indicated that lacZ gene was expressed in the livers. Variations of the electropulsation parameters, including site of electropulsation, wave type, amplitude of the pulses, repetition rate, duty cycle, and duration of electropulsation treatment, do not reveal any statistically significant effects of differences of these variables on the transgene expression. Comparison of the results obtained from direct injection of PLL/pCMVβ (1:1) followed by electropulsation at the LSRPs with that of direct PLL/pCMVβ (1:1) injection, however, suggests that gene transfer by electropulsation achieved a higher transfection efficiency in vivo (p >. 05).

Calcium phosphate (CaPi) precipitation method has been used for decades in gene transfection. The underlying mechanism of this technique has been obscure. In the present study, electrochemical approaches were employed to elucidate the surface ionization and complexation phenomena in this colloidal dispersion. Potentiometric and conductometric titrations were carried out to characterize the ionizable CaPi colloid surfaces. The end points were determined to be at pH 4.32 and 9.88, respectively. Surface charge titration and zeta potential measurements were employed to estimate the point of zero charge (pzc) and isoelectric point (iep) of the CaPi precipitates. The pzc and iep of the CaPi precipitates were found to be at pH 7.09 and pH 7.0, respectively. Addition of pPyMT-1 and pSV2neo plasmid DNA results in a shift of both pzc and iep to higher values of 7.18 and 7.15, respectively. H(+) and OH(-) are the potential determining ions, which are shown to play important roles in determining the magnitude of the surface charge by their reactions with surface sites of CaPi.

The pH of the transfection medium has been shown to be crucial for the calcium phosphate (CaPi)-mediated DNA transfection. This study was undertaken in an attempt to examine several physicochemical properties associated with the pH change as related to DNA transfection efficiency. The zeta potential, flocculation coefficient, and adsorption kinetics of [(3)H]DNA-CaPi complexes onto FR3T3 cells were experimentally examined as a function of pH of the Hepes-buffered saline (HBS) in which the coprecipitates were formed, which were then correlated with the DNA transfection efficiency. (3)H-labeled plasmid DNA was found to be adsorbed on the flocculated CaPi particles for the HBS pH greater than 6.16. Adsorption of CaPi-DNA complexes onto the cell surfaces renders the cell surface charge less negative. Both zeta potentials and the flocculation coefficients of the CaPi-DNA coprecipitates were shown to vary with pH of the system, but no significant difference in the kinetics of adsorption was observed for the CaPi-DNA coprecipitates prepared at various pH. Zeta potentials of the CaPi-DNA coprecipitates lying between 11 and 21 mV were determined to be efficient for the CaPi-DNA complexes to be effectively internalized by the cells. The pH appears to be a determinant factor in the properties studied. The physicochemical properties of the CaPi-DNA complexes play important roles in the interaction between CaPi-DNA complexes and cells, which consequently affect the cell transformation efficiency.

K562 cell line, established from a patient in the blast crisis of chronic myeloid leukemia, expresses high levels of c-myc and bcr/abl gene products. Exposure of K562 cells to 5-fluorouracil (5-FU) resulted in a marked benzidine-positive erythroid differentiation with concomitant reduction in cell proliferation. No change in c-myc mRNA or protein levels occurred during 96 h of drug treatment. In contrast, a biphasic change of p210bcr/abl and the abl-associated kinase activities was observed upon treatment with 5-FU. The change in p210bcr/abl expression may be mediated at the translational level, since the steady-state level and the enzymatic activity of p210bcr/abl are reduced, whereas bcr/abl mRNA levels are unaltered. The results are consistent with the existence of pleiotropic differentiation pathways in K562 cells.

Interaction between polyoma virus (Py) encoded middle tumor antigen (MTAg) and the cellular src gene product, pp60c-src, has been shown to be correlated with the enhancement of pp60c-src kinase activity and the transforming ability of the virus. In the present study, FR3T3 cells were transfected with plasmids encoding MTAg and used to study the effects of the antineoplastic agent, doxorubicin (DOX), on pp60c-src kinase activity. Our results showed that transfection of FR3T3 cells with MTAg results in relatively high levels of MTAg expression and enhancement of pp60c-src kinase activity several folds over that observed in the control cells. Treatment of the transformed MTF4 cells with DOX up to 5 x 10(-6) M was shown to have no effect on the in vitro phosphorylation of MTAg and pp60c-src in the drug-treated cell lysates. On the contrary, treatment of src immunocomplexes with DOX resulted in a reduction in kinase activity. The inhibition appears to be a dose-dependent competition with respect to ATP concentrations.

Insulin and insulin-like growth factor (IGF)-I are thought to be major metabolic regulators of IGF-binding protein-2 (IGFBP-2). We have examined the regulation of IGFBP-2 expression by IGF-I and insulin in 293 cells, a cell line derived from human embryonic kidney. The predominant 34 kDa IGFBP in media conditioned by unstimulated 293 cells was identified as IGFBP-2 by immunoprecipitation. IGFBP-2 levels were increased 6 to 7-fold following incubation with IGF-I, IGF-II or insulin for 48 h. A corresponding increase in IGFBP-2 mRNA was not observed, suggesting that regulation occurred at the translational or post-translational level. The stimulation of IGFBP-2 by IGF-I and insulin was reversibly abolished by incubation with protein synthesis inhibitors such as cycloheximide. Biosynthetic labeling of quiescent 293 cells using [35S]cysteine indicated that incubation with insulin or IGF-I for 24 h increased the synthesis of total cell proteins (predominantly intracellular) and IGFBP-2 (predominantly secreted) to a similar extent (2- to 4-fold). These results suggest that the increase in IGFBP-2 secreted by 293 cells after incubation with IGF-I or insulin largely results from a general stimulation of protein synthesis.

The trimethylsilylation of testosterone and epitestosterone was discussed in detail in this report. Both derivative conditions under which testosterone and epi-testosterone were prepared into TMS-derivatives in the presence of mercaptoethanol as an antioxidizing agent and method for the analysis of the ratio of testosterone to epi-testosterone in urine, based on GC-MS, had been established. The conditions of detection were: carrier gas was helium, derivatives were separated with SE-54 fused silica capillary column, using temperature program and detected by using multiple ion detection mode in which the ion of m/z 432 was the monitoring ion. The method is rapid, sensitive and specific. For the ratio of testosterone to epi-testosterone (testosterone: 20 ng/microliters), there is a linearity between ratio 1:1 and 10:1 (r = 0.998), the limit of detection for testosterone and epi-testosterone is 1 ng, and the minimum concentration of detection in urine is 8 ng/ml.

The insulin-like growth factor-binding proteins (IGFBPs) are thought to determine the distribution of IGF-I and IGF-II between the blood and tissue compartments and to modulate their biological activities. A dynamic metabolic role for one of the IGFBPs, IGFBP-1, is suggested by the fact that plasma IGFBP-1 was increased after fasting and diabetes and rapidly decreased by refeeding or insulin treatment, respectively. IGFBP-1 mRNA also is increased in the livers of diabetic rats and decreased by insulin treatment. To understand the molecular basis for this regulation, we have examined the effects of insulin on IGFBP-1 and IGFBP-1 mRNA in the H4-II-E cell line derived from the well differentiated H35 rat hepatoma. IGFBP-1, identified by ligand blotting and immunoblotting, is the major IGFBP in H4-II-E cells. Incubation of H4-II-E cells with insulin for 24 h decreased IGFBP-1 in the culture medium by approximately 50%. Inhibition was observed at physiological concentrations of insulin (ED50, less than 0.5 nM), but not at higher concentrations of IGF-II. These results, together with the fact that H4-II-E cells do not possess IGF-I receptors with which insulin might cross-react, suggest that insulin acts via the insulin receptor. Insulin inhibited IGFBP-1 in the medium by 80% in the absence of glucose, suggesting that the inhibition is a direct effect of insulin; glucose exerted a smaller independent effect in the absence of insulin. Insulin decreased IGFBP-1 mRNA in H4-II-E cells by 50% within 1 h and by 90% after 2-12 h of incubation. Nuclear run-on transcription assays indicated a corresponding decrease in the rate of IGFBP-1 gene transcription. Pretreatment of H4-II-E cells with dexamethasone stimulated IGFBP-1 transcription and increased steady state IGFBP-1 mRNA; stimulation was abolished by insulin treatment, indicating that inhibition by insulin was dominant over induction by dexamethasone. Thus, insulin, acting through the insulin receptor, rapidly decreases the abundance of IGFBP-1 mRNA in H4-II-E cells. Regulation occurs at least in part at the level of gene transcription. We propose that regulation of IGFBP-1 synthesis is an important component of the regulation of IGFBP-1 by insulin in vivo.

Apolipoprotein (apo) E, a major protein component of plasma lipoproteins, is a physiological ligand for the low density lipoprotein (LDL) receptor as well as for a specific apoE receptor; it is therefore an important modulator of lipoprotein metabolism. In this study we cloned and sequenced bovine apoE complementary DNA. Comparison of nucleotide substitution rates shows that apoE is less conservative than apoA-I and evolves about 30% faster than an average mammalian protein. Although apoE is not a conservative protein, several regions have been well conserved among all eight mammalian sequences now available. These include a 33-amino-acid block immediately upsteam from the third intron/exon junction and the LDL receptor binding region. We have also compared published apoC-I and apoC-II sequences. Both proteins are less conservative than apoE. In particular, apoC-I shows no well-conserved region except for a small region in the common 33-amino-acid block, suggesting that the function of apoC-I does not have stringent structural requirements. On the other hand, in apoC-II the region encoded by exon 4, which consists of the last 29 amino acids of the polypeptide, has been rather well conserved, probably because this region is important for the activation of lipoprotein lipase and chylomicron and very low density lipoprotein metabolism.