The liquid Kangfuxin (KFX) has efficient antifungal activity and can be used in the treatment of vulvovaginal candidiasis in mice.

Vulvovaginal candidiasis (VVC) is an infectious disease caused mainly by Candida albicans. Kangfuxin (KFX) is a traditional Chinese medicine preparation made from Periplaneta americana extracts, which promotes wound healing and enhances body immunity and also acts as an antifungal agent. Here, we evaluated the effect of KFX in the treatment of VVC in vitro and in vivo. The minimum inhibitory concentration (MIC50 ) of KFX against C. albicans ranged from 7·65 to 20·57%. In addition, KFX was more efficient than fluconazole (FLC) in inhibiting the drug-resistant C. albicans, and the effect was more intense after 8 h. The KFX treatment also exhibited good activity in vivo. It restored the body weight and reduced the vulvovaginal symptoms in mice induced with VVC. It downregulated the expression of the hyphae-related gene, HWP1, thus inhibiting the growth and development of C. albicans hyphae. It also increased the number of neutrophils and promoted the secretion of interleukin-17A (IL-17A); however, the levels of interleukin-8 (IL-8) and interleukin-1ß (IL-1ß) decreased in mice with VVC. We deduce that KFX effectively treats vaginal candidiasis in two ways: by inhibiting the growth and development of mycelia to reduce colonization of C. albicans and by promoting the secretion and release of IL-17A and neutrophils in high numbers to fight C. albicans infection. This study provides a theoretical basis for the use of KFX for the clinical treatment of VVC.

[Outcome of pediatric-to-adult liver transplantation:a single-center study in China].

Objective: To explore the outcome of the pediatric-to-adult liver transplantation, including postoperative complications and relevant factors which affecting graft survival. Methods: Data of 55 patients undergoing pediatric-to-adult liver transplantation at the First Affiliated Hospital of Zhejiang University between January 2015 and August 2021 were retrospectively analyzed. The donors consisted of 34 males and 21 females, and the age was (11.8±4.7) years (range: 1 to 17 years). Among the cases,17 cases (30.9%) were donation of brain death,32 cases (58.2%) were donation of cardiac death, and 6 cases (10.9%) were donation after brain death plus cardiac death. The recipients consisted of 32 males and 23 females, and the age was (51.6±10.1) years (range: 27 to 70 years). Among the recipients,10 cases (18.2%) were ABO-incompatible liver transplantation.The influencing factors of early graft survival were analyzed by Student t test,Mann-Whitney U test or χ2 test,respectively.Survival curve was drawn by Kaplan-Meier method.Logistic multivariate analysis was used to analyze the independent relevant factors of early postoperative graft loss. Results: Up to October 31,2021,the follow-up time (M(IQR)) was 36.0(43.1)months(range:5.9 to 81.7 months).There were 13 cases with graft loss (two of them underwent re-transplantation due to acute liver failure).The monofactor analysis indicated that cold ischemia time and donor-recipient blood group matching were the relevant factors affecting the early graft survival rate(both P<0.05).Logistic multivariate analysis showed that cold ischemia time and history of recipient gastrointestinal bleeding were independent relevant factors(both P<0.05).Postoperative hepatic artery thrombosis occurred in 3 cases(5.5%), portal vein thrombosis diagnosed in 4 cases(7.3%), portal vein stenosis occurred in 2 cases(3.6%),biliary complications diagnosed in 7 cases(12.7%), and small liver syndrome was found in 8 cases(14.5%). Conclusions: Adult liver transplantation with pediatric donor liver is an effective method to treat end-stage liver disease.Cold ischemia time and history of recipient gastrointestinal bleeding were independent relevant factors for the early graft survival.

Conversion efficiency of multi-keV L-shell-band X-ray emission.

This study explored the influence of foil thickness, laser pulse width, and laser intensity to optimize the multi-keV X-ray conversion efficiency of a sandwiched (CH/Sn/CH) planar target under laser irradiation at the Shenguang II laser facility. The X-ray photon field values were measured using a set of elliptically bent crystal spectrometers and the conversion efficiencies (ξx) of photon energies were in the range of 3.7-4.3 keV. The experimental results indicate that the X-ray yields of 3.7 to 4.3 keV radiation strongly depend on the laser pulse width, target thickness, and laser intensity. The results also demonstrate that three-layer thin foils can provide an efficient multi-keV X-ray source because they can change the distribution of emitted multi-keV X-rays and target dynamics versus nanosecond laser pulses to produce large, hot, and underdense plasma. However, the underdense plasma produced as a rarefaction wave causes the overdense plasma generated by the laser pulse to expand. Therefore, the laser parameters and foil thickness must be carefully optimized to produce an efficient 3.7 to 4.3 keV X-ray source. Otherwise, the rarefaction waves from both sides of the thin foil may suppress multi-keV X-ray emission. This study represents an important advancement in the development of an efficient multi-keV L-shell-band X-ray source.

[Effects of different fluid resuscitation on renal function and glycocalyx in septic shock rats].

To investigate the effects of different fluid resuscitation on renal function and glycocalyx in septic shock rats. The septic shock SD rats (induced by lipopolysaccharide) were randomly divided into control group, normal saline resuscitation group, Lactate Ringer's solution resuscitation group and succinyl gelatin resuscitation group (n=6 each). The mean arterial pressure, heart rate, serum creatinine (Scr), urea nitrogen (BUN), renal blood flow, renal tissue pathology, serum interleukin-6, tumor necrosis factor α and glycocalyx were measured at baseline, the development of septic shock, 0 hour and 8 hours after resuscitation. When shock was established, the renal blood flow significantly decreased (P<0.05) and recovered to normal at 0 hour and 8 hours after resuscitation. Compared with normal saline group and succinyl gelatin group, the levels of Scr, BUN and glycocalyx components (heparan sulfate, syndecan-1) were significant lower in Lactate Ringer's solution group (P<0.05). The pathology of kidney tissue suggested that the microscopic ischemic damage with Lactate Ringer's solution were minor than the other two groups. Compared with normal saline and succinyl gelatin, Lactate Ringer's solution can reduce the damage of renal function and glycocalyx in septic shock rats.

[Follow-up study of patients with chronic obstructive pulmonary disease complicated with pulmonary hypertension].

Objective: To investigate the relationship between clinical characteristics of patients with chronic obstructive pulmonary disease (COPD) with pulmonary hypertension (PH) and COPD exacerbation over a three-year observation period. Methods: A total of 366 cases of stable COPD patients were enrolled from the Department of Respiratory Medicine of the First Affiliated Hospital of Henan University of Science and Technology. The patients underwent pulmonary function tests(PFT), COPD assessment test (CAT), Saint George's respiratory questionnaire (SGRQ), transthoracic echocardiogrophy(TTE), chest CT and biochemical detection. The likelihood of PH was evaluated based on the peak tricuspid regurgitation velocity (TRV) measured by echocardiography at rest and other indicators, which were represented by low, medium, and high probability, respectively. Highly probability was defined as PH. The mean values of the groups were compared using a two-tailed unpaired t test for normally distributed variables. Qualitative data were assessed using a χ2 test. Pearson correlation analysis was performed, and risk factors were analyzed using logistic regression analysis and stepwise regression analysis. P<0.05 was considered to indicate statistical significance. Results: The prevalence of patients with high likelihood of PH was 18.3% (n=67) in a series of 366 patients with COPD. The median estimated systolic pulmonary artery pressure in patients with PH was (51.7±6.7) mmHg(1mmHg=0.133 kPa). There were differences between patients with high likelihood of PH and those with low to moderate likelihood of PH for the following factors: age (76.0 vs. 64.0), body mass index (BMI) [(21.4±6.0) kg/m2 vs. (22.6±7.2)kg/m2], brain natriureticpeptide (BNP) [(50.8±9.1) pg/ml vs. (36.4±8.1) pg/ml], toral number of acute exacerbation in three-year [(6.1±0.1) times vs. (2.8±0.4) times], CAT (17.0 vs. 10.0), SGRQ (48.9 vs. 32.1), carbon monoxide diffusion percentage of predicted value (DLCO%) [(51.9±21.9)% vs. (67.0±22.1)%]; all the differences being statistically significant(mean P<0.05).There was a negative correlation between DLCO% and SPAP (r=-0.28, P<0.01).In patients with high likelihood of PH, the percentage of low attenuation area (LAA%) and interstitial abnormalities in chest CT were higher than those in patients with low to moderate likelihood of PH (56.1% vs. 34.3% and 30.8% vs. 15.6%, mean P<0.05).LAA% ≥ 30% and pulmonary interstitial abnormalities were independent risk factors for pH [beta value were 1.479, 1.065, OR value was (3.640-5.720), 95%CI (1.462-8.571), mean P<0.01]. The ratio of main pulmonary diameter to aortic artery diameter was significantly correlated with estimated systolic pulmonary artery pressure(r=-0.35, P<0.01).Age ≥75 years, FEV1%predicted value<50% and the presence of PH increased the likelihood of exacerbations of COPD over three years[beta value (0.459-1.211), OR value (3.643-5.722), 95%CI (1.463-8.904), mean P<0.01]. Conclusions: COPD patients with high likelihood of PH assessed by echocardiography were older, had a lower BMI, and presented with a worse health status compared to those with low to moderate likelihood of PH. The presence of PH assessed by echocardiography was related to future COPD exacerbations in COPD patients, and emphysema was closely related to PH assessed by echocardiography.

[Application of the Virtual Reality-Pattern Visual Evoked Potential in Forensic Visual Acuity Evaluation].

ABSTRACT: Objective To explore a kind of visual evoked potential test equipment and method that is more suitable for the application of forensic clinical visual acuity evaluation. Methods Thirty-four volunteers （68 eyes） were selected, including 15 males and 19 females, aged between 20 and 40 years. Test lenses were placed before the tested eyes of volunteers to induce refractive myopia with insert method, and the diopter lenses were adjusted so that the visual acuity level of one eye of volunteers was above 0.8, and the visual acuity of the other eye was at moderate damage level （<0.3 and ≥0.1）. The tests were carried out under the binocular simultaneous asynchronous stimulation mode （hereinafter referred to as "binocular mode"） and monocular separate stimulation mode （hereinafter referred to as "monocular mode"） of virtual reality-pattern visual evoked potential （VR-PVEP）, and the amplitude of PVEP of volunteers under the two modes was compared at four spatial frequencies of 8×8, 16×16, 24×24 and 32×32. Results The differences in the amplitude of P100 wave between monocular and binocular modes at 8×8 spatial frequency had no statistical significance and the differences in amplitude of P100 wave between monocular and binocular modes at 16×16, 24×24, and 32×32 spatial frequencies had statistical significance （P<0.05）. The amplitude of the same eye in monocular mode was higher than that in binocular mode. Through correlation analysis, it was found that the amplitude of P100 wave in monocular mode was moderately correlated with amplitude of P100 wave in binocular mode. Conclusion In forensic identification practice, VR-PVEP is helpful for overcoming the disturbance of poor fixation, and to increase the reliability of PVEP evaluation results. It can greatly shorten the detection time of PVEP and improve work efficiency.

Effects of acarbose and siglitine on blood glucose fluctuation and islet ß-cell function in patients with type 2 diabetes mellitus.

The effects of acarbose and sitagliptin on blood glucose fluctuation and islet ß-cell function in patients with type 2 diabetes mellitus (T2DM) were studied. One hundred and three patients with poorly controlled T2DM with insulin aspart 30 were selected and randomly divided into three groups: group A [continuous subcutaneous insulin infusion (CSII) treatment group], group B (CSII combined with acarbose treatment), group C (CSII combined with sitagliptin treatment). The treatment lasted for two weeks and the clinical indicators in the three groups were measured. The insulin dosage was adjusted according to the blood glucose statuses of the three groups of patients. In the final three days, 72 h of continuous glucose monitoring (CGM) were carried out, and the OGTT test was performed again. The results showed that the MODD (absolute means of daily difference), intra-day blood glucose fluctuation indices [(24 h MBG (mean blood glucose), LAGE (largest amplitude of glycemic excursions) and MAGE (average blood glucose fluctuation)] and postprandial blood glucose fluctuation indices [PGS (postprandial glucose spike), △t, PPGE (postprandial glucose excursion) and T (time) total] in group C and group B were significantly lower than those in group A. Compared with group B, the difference in blood glucose fluctuation indices in group C was not statistically significant (P>0.05). The HOMA-islet (homeostasis model assessment of islet) (CP-DM) index and FC-P (Fasting c-peptide) levels in group C and group B were significantly higher than those in group A (P less than 0.01). The HOMA-IR (CP) index of groups B and C was significantly lower than that of group A (P less than 0.01), and there was no statistically significant difference between groups B and C (P less than 0.05). Sitagliptin combined with intensive insulin pump therapy can reduce blood glucose fluctuation throughout the day, reduce insulin dosage, improve islet B cell function and reduce hypoglycemia better than intensive insulin pump therapy alone.

[Effect of transforming growth factor-ß1 on HBV replication and antigen synthesis in HepG2.2.15 cells with steatosis].

Objective: To investigate the effect of transforming growth factor-ß1 (TGF-ß1) on HBV replication and protein expression in HepG2.2.15 cells with steatosis, as well as the association of TGF-ß1 with suppressor of cytokine signaling-3 (SOCS-3) mRNA and sterol regulatory element-binding protein-1c (SREBP-1c) mRNA during the steatosis of HepG2.2.15 cells. Methods: The cells were divided into HepG2/HepG2.2.15 cell control groups (C1/C2 groups) and HepG2/HepG2.2.15 cell steatosis groups (F1/F2 groups). 5 ng/ml TGF-ß1 was added to the two cell systems for intervention to establish TGF-ß1 intervention groups (T1/T2 groups) and steatosis+TGF-ß1 intervention groups (TF1/TF2 groups). A time-resolved fluorescence analyzer was used to measure HBsAg and HBeAg, and quantitative real-time PCR was used to measure HBV DNA, SOCS-3 mRNA, and SREBP-1 mRNA. A one-way analysis of variance and a factorial analysis were used for the statistical analysis of data. Results: TGF-ß1 significantly reduced the level of HBeAg in C2 group (P = 0.034) and the levels of HBsAg (P < 0.001) and HBeAg (P = 0.004) in F2 group. There was an interaction between steatosis and TGF-ß1 in inhibiting HBsAg. In addition, TGF-ß1 significantly reduced the mRNA expression of SOCS-3 in C1, F1, C2, and F2 groups (P < 0.05) and significantly increased the mRNA expression of SREBP-1c in C1, F1, C2, and F2 groups (P < 0.05), suggesting that there was an interaction between steatosis and TGF-ß1 in downregulating the mRNA expression of SOCS-3 and upregulating the mRNA expression of SREBP-1c. Conclusion: TGF-ß1 does not affect HBV duplication in HepG2.2.15 cells and can inhibit the expression of HBsAg and HBeAg. TGF-ß1 can downregulate the mRNA expression of SOCS-3 and upregulate the mRNA expression of SREBP-1c.

Development of three vitrification-based cryopreservation procedures for shoot tips of China's potato.

China is now the largest potato producing country worldwide. The establishment of efficient conservation techniques for potato germplasm is a prerequisite for breeding of elite cultivars. Potato viral diseases have been a great threat for sustainable potato production in China. Use of virus-free seed tubers is an effective and practical means to control viral diseases. In the present study, three vitrification-based cryopreservation techniques, i.e. droplet-vitrification, encapsulation-vitrification and vitrification were successfully developed for the first time for China's potato. Cultivar 'Zihuabai' was used to optimize key parameters involved in the three vitrification-based procedures. With the optimized parameters, shoot regrowth percentages of 71%, 76% and 43% were obtained for droplet-vitrification, encapsulation-vitrification and vitrification, respectively. The three protocols developed were further tested with eight China's major cultivars, with average shoot regrowth of 61%, 38% and 28% for droplet-vitrification, encapsulation-vitrification and vitrification, respectively. Successful development of the three cryopreservation procedures using a single cultivar will facilitate a number of comparative studies such as cryo-injury, regrowth pattern, genetic stability and efficiency of virus elimination. Testing these three cryogenic procedures for potato major cultivars representing a wide range of genetic background, will help the establishment of potato cryobanking in China and the production of virus-free plants.

Effects of comprehensive rehabilitation on complications and long-term prognoses of patients in vegetative and minimal consciousness states.

OBJECTIVE: To understand the clinical characteristics, prognoses, and prognostic factors of vegetative state (VS) and minimal consciousness state (MCS) patients hospitalized for comprehensive rehabilitation. MATERIALS AND METHODS: The data of VS and MCS patients admitted in the past six years were retrospectively analyzed. Age of onset, etiology, course of disease, complications, and retention of tracheal intubation, gastric tube, and urethral catheter were recorded. All patients were followed for one year after onset, and their Glasgow Outcome Scale (GOS) scores were assessed. Long-term prognostic factors were analyzed using logistic regression. RESULTS: The proportion of patients with traumatic brain injuries in the MCS group was higher than in the VS group (p<0.05), while hypoxic-ischemic encephalopathy accounted for a higher proportion in the VS group; the differences were statistically significant between the two groups. The occurrence of infection was the highest in both groups, followed by spasticity and epilepsy. The occurrence of other complications was slightly different between the two groups but did not reach statistical significance (p>0.05). Age, etiology, and consciousness at admission had a significant impact on the long-term prognoses of VS or MCS patients (p<0.05), and the age of onset was the most important factor (the highest OR). CONCLUSIONS: Infection, spasticity, and epilepsy are common complications in both VS and MCS patients. Recovery of consciousness after one year was shown to be affected by age, etiology, and consciousness at admission.

Lens epithelial cell apoptosis appears to be a common cellular basis for non-congenital cataract development in humans and animals.

Cataract is a major ocular disease that causes blindness in many developing countries of the world. It is well established that various factors such as oxidative stress, UV, and other toxic agents can induce both in vivo and in vitro cataract formation. However, a common cellular basis for this induction has not been previously recognized. The present study of lens epithelial cell viability suggests such a general mechanism. When lens epithelial cells from a group of 20 cataract patients 12 to 94 years old were analyzed by terminal deoxynucleotidyl transferase (TdT) labeling and DNA fragmentation assays, it was found that all of these patients had apoptotic epithelial cells ranging from 4.4 to 41.8%. By contrast, in eight normal human lenses of comparable age, very few apoptotic epithelial cells were observed. We suggest that cataract patients may have deficient defense systems against factors such as oxidative stress and UV at the onset of the disease. Such stress can trigger lens epithelial cell apoptosis that then may initiate cataract development. To test this hypothesis, it is also demonstrated here that hydrogen peroxide at concentrations previously found in some cataract patients induces both lens epithelial cell apoptosis and cortical opacity. Moreover, the temporal and spatial distribution of induced apoptotic lens epithelial cells precedes development of lens opacification. These results suggest that lens epithelial cell apoptosis may be a common cellular basis for initiation of noncongenital cataract formation.

Effects of heat exposure on muscle oxidation and protein functionalities of pectoralis majors in broilers.

This study was conducted to determine the effects of heat stress on pH, lipid peroxidation, protein oxidation, and functionalities of breast meat in broilers. The experimental birds were exposed to 2 temperatures: 24 degrees C (control) and 41 degrees C (heat-stressed) for 0, 1, 2, 3, and 5 h. As compared with control, the pH determinations after slaughter for 30 min and 24 h of chilling, respectively, of the pectoralis major in the heat-stressed groups were significantly lower (P < 0.05). Oxidative stability of i.m. lipid in 3- and 5-h groups (P < 0.01) and protein in 2-, 3-, and 5-h groups (P < 0.01) increased. Protein solubility of pectorilas major decreased after heat exposure. Drip loss and cooking loss were increased in groups from 1 to 5 h of exposure. A reduced strength was noted for myofibrillar protein gels of heat-stressed breasts in exposed groups (P < 0.01); also water-holding capacity of the gels was reduced in treatments of 2, 3, and 5 h (P < 0.01). The results demonstrated that preslaughter exposures to heat reduced the oxidative stability of broiler muscle protein, which might be responsible for decreased protein functionalities such as gelation.

Elliptically bent crystal x-ray spectrometer for time-resolved laser plasma experiments.

Measuring time-resolved spectra is crucial in inertial confinement fusion and radiation source development experiments. An elliptically bent crystal spectrometer is designed to measure X-rays in the range of 2.5-11.0 keV, which was achieved using four different lattice spacings of 0.8512, 0.6687, 0.4246, and 0.2749 nm with spectral resolution E/δE of ∼500. The X-rays emitted from a source at one focus of the ellipse undergo Bragg reflection off a crystal and pass through the second focus of the ellipse to a streak camera slit with 18-mm length and 80-µm width to generate a time-resolved spectrum. An alignment method for the time-resolved spectrometer was developed with the straight line connecting the centers of the two small holes on the fabricated substrate being the axis of the ellipse, thus allowing the spacing between the source and the elliptical crystal to be tuned to couple with the streak camera. The time-resolved spectrometer's performance was experimentally tested at the Shenguang II laser facility. The results indicate that its performance is close to that predicted theoretically.

Alterations of elastin and elastase-like activities in aortae of diabetic rats.

The elastin content of the aortic muscle and the elastase-like activity of the extracts of aortic muscle were studied in spontaneously diabetic BB rats and in rats made diabetic by a single bolus i.v. injection of alloxan. In both modes of diabetes, the total alkaline-insoluble aortic elastin content was significantly reduced in diabetic rats compared to that in the corresponding control rats. This reduction in aortic elastin was also accompanied by a consistent increase in the elastase-like activities of the aortic extracts prepared from the same tissues. Such a reciprocal relationship between aortic elastin content and elastase-like activity has previously been reported in rats with malignant hypertension. Since the rats used in this study were not hypertensive, the altered elastin metabolism observed in this work is likely to be a manifestation of diabetic disease and may in part account for the vascular changes associated with diabetes mellitus.

Smoking prevalence and its influence on disease course and surgery in Crohn's disease and ulcerative colitis.

BACKGROUND: Smoking demonstrates divergent effects in Crohn's disease (CD) and ulcerative colitis (UC). Smoking frequency is greater in CD and deleterious to its disease course. Conversely, UC is primarily a disease of nonsmokers and ex-smokers, with reports of disease amelioration in active smoking. AIM: To determine the prevalence of smoking and its effects on disease progression and surgery in a well-characterised cohort of inflammatory bowel diseases (IBD) patients. METHODS: Patients with smoking data of the Sydney IBD Cohort were included. Demographic, phenotypic, medical, surgical and hospitalisation data were analysed and reported on the basis of patient smoking status. RESULTS: 1203 IBD patients were identified comprising 626 CD and 557 UC with 6725 and 6672 patient-years of follow-up, respectively. CD patients were more likely to smoke than UC patients (19.2% vs. 10.2%, P < 0.001). A history of smoking in CD was associated with an increased proportional surgery rate (45.8% vs. 37.8%, P = 0.045), requirement for IBD-related hospitalisation (P = 0.009) and incidence of peripheral arthritis (29.8% vs. 22.0%, P = 0.027). Current smokers with UC demonstrated reduced corticosteroid utilisation (24.1% vs. 37.5%, P = 0.045), yet no reduction in the rates of colectomy (3.4% vs. 6.6%, P = 0.34) or hospital admission (P = 0.25) relative to nonsmokers. Ex-smokers with UC required proportionately greater immunosuppressive (36.2% vs. 26.3%, P = 0.041) and corticosteroid (43.7% vs. 34.5%, P = 0.078) therapies compared with current and never smokers. CONCLUSIONS: This study confirms the detrimental effects of smoking in CD, yet failed to demonstrate substantial benefit from smoking in UC. These data should encourage all patients with IBD to quit smoking.

Structural analysis of templates and RNA polymerase III transcripts of Alu family sequences interspersed among the human beta-like globin genes.

Cloned DNA fragments form the human beta-like globin genomic region can be transcribed in vitro by RNA polymerase III. We have investigated the structure of two templates and their transcripts by DNA sequencing, size fractionation of ribonuclease T1 generated oligonucleotides, and ribonuclease H digestion of RNA : DNA duplexes. The data indicate the repetitive DNA sequences, members of the Alu family of interpersed 300 bp reiterated DNA, are imbedded in both templates. The RNAs transcribed from them are composed of an entire Alu family sequence at their 5' ends linked to 3' ends of non-repetitive sequence.

The aqueous humor is capable of generating and degrading H2O2.

PURPOSE: To determine the ability of the aqueous humor to generate and degrade H2O2, the effect of environmental factors such as oxygen tension and temperature, and the constituents contributing to the observed results. METHODS: Aqueous humor was carefully removed from bovine eyes within 3 hours of death. Standard and new techniques were used to follow H2O2 degradation and formation. Catalase activity was measured by H2O2 decomposition, usually at 100 microM and 10 mM H2O2, and in some cases by O2 generation. RESULTS: Bovine aqueous humor generated and degraded H2O2. The generation of H2O2 was minimal at 0 degrees C but increased as temperature increased, so that at 37 degrees C at 3 hours, approximately 90 microM H2O2 was observed. Degradation was more complex. At 0 degrees C, only a slow rate of degradation was observed. At 25 degrees C, it was more rapid, and a steady state between generation and degradation was observed at approximately 30 microM. However, at 37 degrees C, starting with 100 microM H2O2, degradation was initially rapid, but then generation became predominant and H2O2, concentration increased to more than 100 microM in 3 hours. No H2O2 was generated in the absence of O2, and H2O2 production increased with increasing O2 tension. Both low and high molecular weight components contributed to the degradation, but synthesis was completely dependent on low molecular weight constituents. Ascorbic acid and metal ions such as Cu+ made a major contribution to H2O2 production. Catalase may be the macromolecular component responsible for aqueous H2O2 decay, as evidenced by H2O2 degradation, inhibition by boiling or 3-aminotriazole, and the approximate correspondence between oxygen generation and H2O2 degradation. CONCLUSIONS: The results indicate that the aqueous humor is capable of producing levels of H2O2 that have been shown previously to cause cataract in organ culture. Changes in aqueous humor metal ion content and concentration of oxygen level have profound effects on H2O2 concentration and may effect lens viability. The variation in published H2O2 levels may in part be the result of the conditions under which the aqueous humor was obtained, stored, and used for assay. The observed steady state H2O2 concentration of 1 microM in fresh aqueous from bovine eyes must be maintained by the metabolism of surrounding tissues as well as intrinsic components capable of degrading H2O2.

Development and characterization of an H2O2-resistant immortal lens epithelial cell line.

PURPOSE: To determine how nature would protect lens epithelial cells from H2O2 stress, an immortal murine lens epithelial cell line, alphaTN4-1, was subjected to gradually increasing H2O2 levels over a period of approximately 8 months. The resultant conditioned cells grew normally when exposed daily to 125 microM H2O2, whereas normal cells died within 9 hours. This communication describes changes in the cell biology of the conditioned cells that allowed them to remain viable. The manner in which critical biochemical parameters were affected in both conditioned and normal cells is also reported. METHODS: Conditioned cells were obtained by gradually increasing the concentration of H2O2 over a period of approximately 8 months, introducing an aliquot of H2O2 every 24 hours. A wide spectra of biological parameters were evaluated, including catalase, GSH peroxidase and other antioxidative enzyme activities, cell number and cell viability, non-protein thiol, ATP, transport systems, thymidine incorporation, and DNA cleavage. RESULTS: Surprisingly, the conditioned cells did not degrade the medium H2O2 more rapidly than normal cells. However, analyses of the antioxidative defenses indicated that catalase activity was increased 60-fold and glutathione peroxidase (GSH Px) approximately 2.7-fold. Glucose-6-phosphate dehydrogenase, GSH S-transferase, and GSSG reductase also had increased activity. Using one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, in situ trypsin digestion and matrix-assisted laser desorption/ionization mass spectrometry, a highly amplified doublet in the conditioned cell preparation was shown to be GSH S-transferase alpha-1 and alpha-2 isomers. Examination of key biochemical parameters indicated that while most such parameters in the conditioned cells showed marked decay in the first hour or so after stress, recovery was then observed and within a few hours, these parameters were back in the normal range. In contrast, damage in the normal cells was not repaired. The damage to DNA was shown to involve Fenton chemistry. In the presence of a metal ion chelator, normal cells survive H2O2 stress. CONCLUSIONS: The overall conclusion from this investigation is that nature has chosen to respond to the H2O2 stress by not only increasing the activity of enzymes degrading H2O2 but also the systems involved in repair, generation of reducing potential, and detoxification. All but one system of those evaluated appears to be permanently modified.

Hydrogen peroxide-induced DNA damage in bovine lens epithelial cells.

The present investigation was undertaken to determine the types and extent of DNA damage resulting from incubation of primary cultures of bovine lens epithelial cells with hydrogen peroxide. Significant numbers of DNA single-strand breaks were detected by alkaline elution after exposure to as little as 25 microM H2O2 for 5 min at 37 degrees C. The extent of single-strand breakage was concentration dependent and linear from 25 to 200 microM H2O2. The observed single-strand breaks appear primarily due to the action of the hydroxyl radical via a Fenton reaction as both an iron chelator, 1,10-phenanthroline and OH. scavengers, including DMSO, KI and glycerol, significantly inhibited the DNA-damaging effect of H2O2. Diethyldithiocarbamate, an inhibitor of superoxide dismutase, further potentiated the DNA-damaging effects of H2O2, presumably by increasing the steady-state concentration of Fe2+. DNA-protein cross-linking was not observed. In addition, significant levels of 5,6-saturated thymine residues or pyrimidine dimers were not detected after modification of the alkaline elution methodology to allow the use of either E. coli endonuclease III or bacteriophage T4 endonuclease V, respectively. No double-strand breaks were detected after incubation of epithelial cell cultures with H2O2 concentrations of up to 400 microM for 10 min and subsequent neutral filter elution. Since, in vivo, the lens epithelium contains populations of both quiescent and dividing cells, the degree of susceptibility to oxidative damage was also studied in actively growing and plateau-phase cultures. Reduced levels of single-strand breakage were observed when plateau-phase cultures were compared to actively growing cells. In contrast, essentially no differences in repair rates were noted at equitoxic doses of H2O2. The above results suggest that lens epithelial cells may be particularly sensitive to oxidative damage and thus are a good model system in which to study the effects of oxidative stress.