Delayed seroreversion of specifical antibody against HIV in HIV-exposed infants: A retrospective cohort study.

OBJECTIVES: To investigate the seroreversion time in HIV-1-exposed but uninfected infants from two tertiary hospitals in China. METHODS: This study retrospectively investigated the data of perinatal, HIV-1-exposed infants from hospitals in Beijing and Shenzhen. Maternal and infant medical records from both hospitals from January 2009 to December 2019 were reviewed, and the HIV antibody seroreversion times of infants were determined. From 2009 to 2019, a total of 485 HIV-1-exposed but uninfected infants were enrolled. The majority of infants were born at term with normal birth weight. RESULTS: The seroreversion rates were 89.3%, 94.2% and 100% at 12, 18 and 24 months of age, respectively. There were no significant associations between seroreversion and several risk factors, such as gender, birth weight, gestational age, mode of delivery, postpartum prophylaxis and antiretroviral treatment duration. The mean value of HIV-specific immunoglobulin G concentration decreased from 15.4 at day 42 to 0.03 after 24 months in HIV-exposed, uninfected infants. CONCLUSIONS: Clearance of HIV antibodies could take more than 18 months in a small number of perinatally exposed infants. Caution should be used in excluding or diagnosing perinatal HIV infection in children with long persistence of HIV antibodies.

[Prevalence and risk factors of ischemic stroke in rural areas of Liaoning province].

Objective: To explore the prevalence and risk factors of ischemic stroke in rural areas of Liaoning province. Methods: The study was a cross-sectional survey. From September 2017 to May 2018, a total of 10 926 rural residents aged ≥40 years were investigated in Chaoyang county, Lingyuan, Liaoyang county and Donggang city of Liaoning province. The investigation included questionnaire survey, physical examination and laboratory examination.Univariate and multivariate logistic regression models were used to analyze the risk factors of ischemic stroke. Results: The prevalence of ischemic stroke in the rural areas of Liaoning province was 5.51% (602/10 926), and the standardized prevalence rate was 4.04%. The standardized prevalence rate of male (5.05%) is higher than that of female (3.44%). The prevalence of ischemic stroke increased with age in both males (P<0.01) and females (P<0.01). Multivariate logistic regression analysis showed that age increase(compared with 40-49 years old group, 50-59 years old, OR=2.08, 95%CI 1.31-3.30, P=0.02; 60-69 years old, OR=3.90, 95%CI 2.51-6.05, P<0.01; 70-79 years old, OR=5.32, 95%CI 3.37-8.34, P<0.01; ≥80 years old, OR=3.64, 95%CI 2.00-6.62, P<0.01), male(OR=2.35, 95%CI 1.95-2.84, P<0.01),family history of stroke(OR=2.18, 95%CI 1.83-2.60, P<0.01),coronary heart disease (OR=2.01, 95%CI 1.52-2.66, P<0.01), hypertension (OR=2.82, 95%CI 2.21-3.60, P<0.01), diabetes mellitus (OR=1.36, 95%CI 1.11-1.67, P=0.03) and overweight/obese (OR=1.22, 95%CI 1.02-1.47, P=0.03) were the major risk factors of ischemic stroke. Conclusions: The prevalence of ischemic stroke in rural areas of Liaoning province is high. Age, male, family history of stroke, coronary heart disease, hypertension, diabetes mellitus, overweight/obesity are the risk factors of ischemic stroke in rural areas of Liaoning province.

[Application of RapidHIT™ 200 System in Forensic Medicine].

OBJECTIVES: To validate the analysis capability of RapidHIT™ 200 system for four kinds of routine forensic samples and the recyclable capability of template, template DNA and PCR products in the process of twice duplicate detection. METHODS: The buccal swabs underwent the test twice by RapidHIT™ 200 system, and the template DNA and PCR products that arose in the system were also tested for two times. After four kinds of routine forensic samples were detected by RapidHIT™ 200 system, the follow-up tests of the template, template DNA and PCR products that arose in the system were performed. RESULTS: The STR loci could be detected in the buccal swabs by the system for the first time. However, part of the STR loci lost during the second test. And the peak value obtained in the second test was significantly reduced than the one in the first time. The average STR loci detection rates of the template DNA and PCR products were both less than 50% in the second test, which were significantly reduced than that in the first test. In addition, the analysis capability of the system for the tissues and buccal swabs was better than that for the blood and cigarette butts. Compared with the first test, the STR loci detection rate of the tested items, template DNA and PCR products decreased with the numbers of tests. CONCLUSIONS: RapidHIT™ 200 system is more effective in retesting buccal swabs than other samples, whereas the items, DNA template, PCR products obtained in the first and second time cannot be directly used for the further application and study of forensic medicine.

Photon-conversion and sensitization evaluation of Eu³âº in a borate glass system.

Photon conversion is exhibited in a borate (LKZBSB) glass system containing Eu(3+), and the enhanced characteristic emissions of Eu(3+) with the codoping of Ce(3+) have been verified. A large Judd-Ofelt intensity parameter Ω2 of Eu(3+) indicates a high asymmetrical and strong covalent environment around rare-earth (RE) ions in LKZBSB glasses and spontaneous emission probability and a maximum emission cross section of the dominant 5D0→7F2 transition were derived to be 370 s(-1) and 1.28×10(-21) cm2, respectively, revealing the potential UV→visible photon-conversion capacity of Eu(3+). Absolutely quantitative evaluation illustrates that Eu(3+) is a favorable photon-conversion center to achieve high photon-conversion efficiency. The addition of Ce(3+) is beneficial to realizing effective red emission of Eu(3+), which possesses commercial value by decreasing the dopant of expensive europium compounds. As an expectation, this photon-conversion LKZBSB glass system can promote the development of a photon downconversion layer for solar cells, which are particularly used in outer space with intense UV radiation.

Prognostic model for survival of local recurrent nasopharyngeal carcinoma with intensity-modulated radiotherapy.

BACKGROUND: Intensity-modulated radiotherapy (IMRT) is the main salvage treatment for advanced locally recurrent nasopharyngeal carcinoma (NPC); however, survival outcomes vary. We aimed to construct a prognostic-score model to identify patients who could benefit from salvage IMRT. METHODS: This retrospective study involved 251 patients with locally recurrent NPC. The following parameters were analysed following IMRT: patient performance status, age, gender, late complications, T-stage of recurrence, synchronous nodal recurrence, primary gross tumour volume (GTV-nx), disease-free interval, re-irradiation dose and chemotherapy. The model was based on the hazard ratio coefficients of six significantly negative prognostic factors for survival. RESULTS: Significantly negative prognostic factors included Karnofsky Performance Status ≤70, age >50 years, late complications, recurrent T(3-4) stage, synchronous nodal recurrence and GTV-nx >30 cm(3). Three subgroups were defined according to model scores: low risk (0-4), intermediate risk (5-8) and high risk (9-15). The 5-year overall survival rates were 64.3%, 32.2% and 7.7%, respectively. The main cause of death was radiation-induced complications. CONCLUSION: The prognostic-score model demonstrated that re-irradiation with IMRT is suitable for low-risk and intermediate-risk patients but may be unsuitable for high-risk patients. Further research into the protection of critical adjacent organs to reduce late complications in these patients is warranted.

Late toxicities after intensity-modulated radiotherapy for nasopharyngeal carcinoma: patient and treatment-related risk factors.

BACKGROUND: The objective of this study is to analyse the factors affecting late toxicity for nasopharyngeal carcinoma (NPC) patients treated with intensity-modulated radiotherapy (IMRT). METHODS: Seven hundred and eighty-nine consecutive NPC patients treated with IMRT at our centre from January 2003 to February 2008 were retrospectively analysed. Radiotherapy-related complications were categorised using the RTOG Late Radiation Morbidity Scoring Criteria and the Common Terminology Criteria for Adverse Events (Version 3.0). Two hundred and thirty-three patients were treated with IMRT alone (group 1) and 556 patients underwent cisplatin-based chemotherapy (group 2). RESULTS: Median follow-up was 65 months (range, 4-106 months). The 5-year major late toxicity rate was significantly greater in group 2 than group 1 (63.2% vs 42.0%, P<0.001). Multivariate analyses showed that N category, T category and chemotherapy were significant factors. The maximal dose (Dmax) to the temporal lobe was a significant factor affecting temporal lobe injury (TLI), with a hazard ratio of 1.26 (95% confidence interval (CI), 1.18-1.35; P<0.001) per 1-Gy increase. The 5-year TLI rate increased from 0.8% for 284 lobes with Dmax <65.77 Gy to 27.1% for 176 lobes with greater doses (P<0.001). Logistic regression showed that the hazard ratio attributed to the parotid gland mean dose was 1.36 (95% CI, 1.21-1.53; P<0.001) per 1-Gy increase. Chemotherapy was not a significant factor (P=0.211). CONCLUSION: With the application of IMRT, the incidence of radiation-related complications has been reduced except for TLI. The significant factors affecting the risk of TLI included T category, chemotherapy and Dmax.

[Association between diabetes prevalence and mortality risk in the elderly aged 60 years and above in Liaoning Province, 2017-2019].

Objective: To investigate the prevalence of diabetes in the elderly aged ≥60 years in Liaoning Province from 2017 to 2019 and analyze the impact of blood glucose control on all-cause mortality and cardiovascular disease (CVD) mortality. Methods: A survey was conducted in the elderly aged ≥60 years in Liaoning from 2017 to 2019 to collect the information about the prevalence of diabetes and other chronic diseases in the diabetes patients. The mortality of the enrolled subjects was investigated in September 2023. Cox proportional hazards regression models were used to estimate the association between blood glucose control in the elderly with diabetes and the risks of all-cause mortality and CVD mortality. Results: The crude prevalence of diabetes in the elderly aged ≥60 years was 20.2% (2 014/9 958) in Liaoning from 2017 to 2019, and the standardized prevalence rate was 19.9%. The prevalence rates of hypertension, dyslipidemia, and overweight/obesity in the diabetes patients were 77.0%, 51.7%, and 67.5% respectively. The median follow-up time was 5.5 years, and the all-cause mortality and CVD mortality rates in the diabetes patients were 244.3/10 000 person-years and 142.9/10 000 person-years, respectively. The results of the Cox proportional hazards regression model analysis showed that compared with non-diabetic individuals, diabetes patients had an increased risk of all-cause mortality by 1.68 times [hazard ratio (HR)=1.68, 95%CI: 1.44-1.94] and an increased risk of CVD mortality by 1.56 times (HR=1.56, 95%CI: 1.29-1.89). The differences in risks of all-cause mortality and CVD mortality between the diabetes patients with normal fasting blood glucose and glycated hemoglobin levels and people without diabetes were not significant (all P>0.05). The failure to meet either the FPG or HbA1c target increased the risk of all-cause mortality (all P<0.05). For individuals who failed to meet the HbA1c target, there was an increased risk of CVD mortality (all P<0.05). Conclusions: The comorbidity rate of chronic diseases was higher in the elderly with diabetes than in the elderly without diabetes in Liaoning. Elderly diabetes patients can benefit from good blood glucose control.

[Effect of subpressure on the bonding strength of resin to polycrystalline particulates modified zirconia ceramic].

Objective: To explore the effect of subpressure on the bonding strength of resin to polycrystalline particulates modified zirconia ceramic. Methods: One hundred and twenty pre-sintered zirconia discs were prepared and divided into the control group, the sandblasting group and the 30, 50, 70 s acid etching group (24 per group) by the random number table method. There was no additional treatment in the control group and sandblasting group before sinering. The 30, 50, and 70 s acid etching groups were immersed in HF for 30, 50, 70 s, respectively, and then they were placed into CaCl2 solution for 90 s and dipped in NaOH solution at 80 ℃ for 2 h. After sintering, the sandblasting group was subjected to sandblasting. The surface tomography and roughness were tested. According to whether subpressure was applied or not after the adhesives were applied, each group was randomly divided into two subgroups with a random number table: a subpressure subgroup and a normal pressure subgroup (12 per subgroup). Resin columns were bonded to these specimens. Shear bonding strength (SBS) test was conducted and the bonding interface, fracture surface and failure mode were analyzed. Results: The surface of control group was smooth, and its roughness was (0.24±0.11) µm. The rough surface was formed after sandblasting in the sandblasting group, and its roughness was (0.95±0.12) µm. The surface roughness of 30, 50, 70 s acid etching groups [(0.60±0.15), (1.04±0.11), (1.57±0.16) µm] increased as the HF immersion time prolonged, and the difference in surface roughness of zirconia specimens among each group was statistically significant (P<0.05). The SBS values between zirconia and resin of all the subpressure subgroups, namely: the control group, the sandblasting group, and the 30, 50, 70 s acid etching group [(13.56±1.19), (20.98±2.11), (17.37±2.44), (24.19±2.97), (21.36±2.16) MPa] were significantly stronger than those in the normal pressure subgroups, namely: the control group, sandblasting group, 30, 50, 70 s acid etching group [(10.74±0.93), (18.47±2.14), (14.81±1.54), (20.74±2.56), (17.75±2.54) MPa] (P<0.05). No obvious gaps and bubbles were observed in the bonding interfaces in subpressure subgroups. The proportion of mixed failure was significantly increased after applying subpressure (P<0.05). Conclusions: The subpressure can effectively enhance the bonding strength between the resin and polycrystalline particulates modified zirconia ceramic and improve the bonding effect.

[Clinical study on the accuracy of occlusal contacts on digital model].

Objective: To explore the accuracy of occlusal contacts on digital model made by intraoral scanner. Methods: Twenty healthy subjects [6 males, 14 females, (24.4±1.4) years old] with intact dentition were randomly recruited from postgraduate students in Capital Medical University School of Stomatology who volunteered to participate in this study. For each participant, the 2nd and 3rd quadrant of natural dentition was scanned. A diagnostic test design was performed. The occlusal contacts of the maximal intercuspal position (MIP) were extracted with the transillumination of silicone interocclusal records, and the extraction threshold was set as ≤50 µm. Intraoral scanning system was used to scan in MIP and generate occlusal contacts on digital model. Five groups were designed as test groups according to included tooth position: group 1 (buccal scanning ranged from tooth 21 to 23), group 2 (buccal scanning ranged from tooth 23 to 26), group 3 (buccal scanning ranged from tooth 24 to 26), group 4 (buccal scanning ranged from tooth 25 to 26), group 5 (buccal scanning ranged from tooth 21 to 26). Five groups occlusal contacts on digital model were generated respectively. According to the relevant literature, the upper occlusal surface was divided into 28 partitions, and the accuracy of occlusal contacts on digital model was calculated with the transillumination of silicone interocclusal records as the reference standard. Subgroup analysis was performed according to anterior teeth area, premolars area and molars area. Results: The accuracy of occlusal contacts on digital models of the half dentition in five buccal scanning positions were: group 1 (86.8%), group 2 (92.0%), group 3 (90.7%), group 4 (91.1%), group 5 (90.4%), and the accuracy of occlusal contacts in group 1 was significantly lower than those in the other four groups (P<0.05). The accuracy of anterior teeth area were 85.6%-93.9%; the accuracy of premolar area were 92.5%-94.4%; the accuracy of molar area were 77.3%-93.6%, group 1 was significantly lower than those in the group 4 in molars area (P<0.05), the accuracy of anterior area was statistically less than premolars area and molars area in group 1 (P<0.05). There was no statistical difference in pairwise comparison between the three sections (P>0.05). Conclusions: The digital models scanned intraoral methods provide accurate, quantitative measures of occlusal contacts when transillumination contacts are the reference standard.

Targeting of HIF-alpha to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation.

Hypoxia-inducible factor (HIF) is a transcriptional complex that plays a central role in the regulation of gene expression by oxygen. In oxygenated and iron replete cells, HIF-alpha subunits are rapidly destroyed by a mechanism that involves ubiquitylation by the von Hippel-Lindau tumor suppressor (pVHL) E3 ligase complex. This process is suppressed by hypoxia and iron chelation, allowing transcriptional activation. Here we show that the interaction between human pVHL and a specific domain of the HIF-1alpha subunit is regulated through hydroxylation of a proline residue (HIF-1alpha P564) by an enzyme we have termed HIF-alpha prolyl-hydroxylase (HIF-PH). An absolute requirement for dioxygen as a cosubstrate and iron as cofactor suggests that HIF-PH functions directly as a cellular oxygen sensor.

Effects of subpressure on the sealing ability of dental sealant in vitro.

OBJECTIVES: Pits and fissures sealing with flowable materials is a popular method for preventing caries in preventive dentistry while there is still microleakage existed. This in vitro study aimed to explore the effects of subpressure technique on the sealing ability of pit and fissure sealant. MATERIALS AND METHODS: One hundred and forty-one extracted human premolars were collected in this study and treated with different pressure (atmosphere pressure as group C, -0.04 MPa as group S4 and -0.08 MPa as group S8). Thermocycling (×5000) was also performed. Penetration percentage, microleakage, cross-sectional microhardness (Knoop, KMH) and mineral loss were evaluated. Kappa tests, Friedman nonparametric and two-way ANOVA were used for data analysis. RESULTS: Penetration percentages of group S4 and S8 were significant higher compared to that of group C. Microleakage of groups was similar before thermocycling, while subpressure groups showed lower scale of microleakage after thermocycling. Data of KMH and mineral loss showed significant differences between subpressure and thermocycling groups. SIGNIFICANCE: Subpressure technique could increase the penetration of pit and fissure sealant, decrease microleakage and increase resistance of demineralization after thermocycling. This novel technique may have great potential for preventing from secondary caries.

Expression of telomerase inhibits hydroxyl radical-induced apoptosis in normal telomerase negative human lung fibroblasts.

In tumor cells telomerase activity is associated with resistance to apoptosis and the introduction of the human telomerase reverse transcriptase (hTERT) subunit into normal human cells is associated with life span extension of the cells. To determine the role of telomerase in regulating apoptosis, telomerase negative human embryo lung fibroblasts were transfected with the hTERT gene. Unlike the control fibroblasts, the telomerase-expressing cells had elongated telomeres and were resistant to apoptosis induced by hydroxyl radicals. The results indicate that expression of telomerase and, thus, the maintenance of telomere length in normal human somatic cells caused resistance to not only cellular senescence but also apoptosis. Moreover, we found that hydroxyl radical-induced apoptosis in telomerase-expressing and control fibroblasts was caspase-3 independent. These findings have revealed a new type of interrelation between telomerase and caspase-3, which may indicate that in this case the expressed telomerase may inhibit apoptosis at a site not related to the caspase-3 cascade.

Protein kinase C in beta-cells: expression of multiple isoforms and involvement in cholinergic stimulation of insulin secretion.

The mammalian protein kinase C (PKC) family consists of at least 11 distinct isotypes with marked differences in tissue distribution, localization, cofactor dependence and substrate specificity. Evidence exists for the expression of some of the PKC isoforms in pancreatic beta-cells but no comprehensive analysis of all the known PKC types has been accomplished. To assess the functional relevance of phosphorylation by PKC in the mechanism of insulin secretion we firstly investigated the expression of PKC isoforms in pancreatic beta-cells. The combination of reverse transcription-polymerase chain reaction (RT-PCR), Northern analysis and immunoblotting demonstrated the expression of PKC-alpha, beta II, epsilon, zeta, lambda and mu in MIN6 beta-cells. PKC-mu has not previously been detected in beta-cells. Expression of PKC-delta was also observed at the mRNA level; however, the protein could not be detected by Western blotting in MIN6 cells but was readily observed in RINm5F beta-cells. In short-term incubations, insulin release from MIN6 cells was augmented by 12-0-tetradecanoyl-phorbol-13-acetate (TPA), by carbachol, and by 40 mM K+. Culture of MIN6 cells overnight with TPA resulted in down-regulation of PKC-alpha (totally) and epsilon (partially), without significant change in the other isoforms. In such TPA-treated cells, the secretory response to TPA and to carbachol was abolished but not that elicited by high K+. It is suggested that PKC-alpha and/or epsilon may play a role in cholinergic potentiation of insulin secretion.

Alteration of dopaminergic markers in gastrointestinal tract of different rodent models of Parkinson's disease.

Parkinson's disease (PD) is a progressive neurological disorder that is often associated with various gastrointestinal (GI) symptoms. The link between the alteration of dopaminergic system and the symptoms of the GI tract in PD is complicated. To determine the changes in the dopaminergic system in the GI tract in PD, two kinds of rodent PD models were used in the present study. One was 6-hydroxydopamine (6-OHDA) -treated rats in which 6-OHDA was microinjected in the bilateral substantia nigra (SN). The other was 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) -treated mice in which MPTP was injected intraperitoneally. Immunofluorescence, reverse transcription (RT)-real time polymerase chain reaction (PCR) and Western blot were used to evaluate and compare the levels of mRNA and protein expression of tyrosine hydroxylase (TH) and dopamine transporter (DAT) in the GI tract between normal and rodent PD models, as well as between 6-OHDA-treated rats and MPTP-treated mice. The results indicated that TH- and DAT-positive cells were widely distributed in the GI tract. There were significant differences in TH and DAT expression in the GI tract between normal and PD models, as well as between 6-OHDA-treated rats and MPTP-treated mice. The protein levels of TH and DAT in the GI tract were significantly increased in 6-OHDA-treated rats, but the protein level of TH was significantly decreased in MPTP-treated mice. In addition, there was visible atrophy of gastric epithelial parietal cells in MPTP-treated mice, although the protein level of DAT was not significantly changed. The different alterations of dopaminergic system in the GI tract of the two kinds of PD models might underline the differences in GI symptoms in PD patients and might be correlated with the disease severity and disease process affecting the GI tract.

Target gene selectivity of hypoxia-inducible factor-alpha in renal cancer cells is conveyed by post-DNA-binding mechanisms.

Inactivation of the von Hippel-Lindau tumour suppressor in renal cell carcinoma (RCC) leads to failure of proteolytic regulation of the alpha subunits of hypoxia-inducible factor (HIF), constitutive upregulation of the HIF complex, and overexpression of HIF target genes. However, recent studies have indicated that in this setting, upregulation of the closely related HIF-alpha isoforms, HIF-1alpha and HIF-2alpha, have contrasting effects on tumour growth, and activate distinct sets of target genes. To pursue these findings, we sought to elucidate the mechanisms underlying target gene selectivity for HIF-1alpha and HIF-2alpha. Using chromatin immunoprecipitation to probe binding to hypoxia response elements in vivo, and expression of chimaeric molecules bearing reciprocal domain exchanges between HIF-1alpha and HIF-2alpha molecules, we show that selective activation of HIF-alpha target gene expression is not dependent on selective DNA-binding at the target locus, but depends on non-equivalent C-terminal portions of these molecules. Our data indicate that post-DNA binding mechanisms that are dissimilar for HIF-1alpha and HIF-2alpha determine target gene selectivity in RCC cells.

Use of novel monoclonal antibodies to determine the expression and distribution of the hypoxia regulatory factors PHD-1, PHD-2, PHD-3 and FIH in normal and neoplastic human tissues.

AIMS: The cellular response to hypoxia includes the hypoxia inducible factor (HIF)-induced transcription of genes involved in diverse processes such as glycolysis, angiogenesis and the growth of experimental tumours. Regulation of the level of hypoxia inducible factors 1alpha and 2alpha (HIF-1alpha and HIF-2alpha) is a primary determinant of HIF activity. Recent biochemical and candidate gene approach studies have led to the discovery of three HIF-regulatory prolyl hydroxylases, PHD-1, -2 and -3 and an asparaginyl hydroxylase, also known as FIH (factor inhibiting HIF). In this study, we raised and characterized monoclonal antibodies against PHD-1, PHD-2, PHD-3 and FIH. METHODS AND RESULTS: Immunohistochemistry of normal tissues with these monoclonal antibodies demonstrated a wide distribution in epithelial cells, stromal cells and leucocytes, with cytoplasmic staining predominating over nuclear staining. A preliminary study of tumours showed variable staining in tumour, stromal and inflammatory cells. While all tumour types showed some positive staining with each antibody, the overall pattern suggested a slight decrease in the amount of staining seen with PHD-1, -2 and -3 and an increase in FIH staining in neoplasia compared with corresponding normal tissues. CONCLUSIONS: These monoclonal antibodies will allow further larger scale studies to determine the significance of PHD and FIH expression in neoplasia.

Oxygen-regulated and transactivating domains in endothelial PAS protein 1: comparison with hypoxia-inducible factor-1alpha.

Endothelial PAS protein 1 (EPAS1) is a basic helix-loop-helix Per-AHR-ARNT-Sim transcription factor related to hypoxia-inducible factor-1alpha (HIF-1alpha). To analyze EPAS1 domains responsible for transactivation and oxygen-regulated function, we constructed chimeric fusions of EPAS1 with a GAL4 DNA binding domain, plus or minus the VP16 activation domain. Two transactivation domains were defined in EPAS1; a C-terminal domain (amino acids 828-870), and a larger internal domain (amino acids 517-682). These activation domains were interspersed by functionally repressive sequences, several of which independently conveyed oxygen-regulated activity. Two types of activity were defined. Sequences lying N-terminal to and overlapping the internal transactivation domain conferred regulated repression on the VP16 transactivator. Sequences lying C-terminal to this internal domain conveyed repression and oxygen-regulated activity on the native EPAS1 C-terminal activation domain, but not the Gal/VP16 fusion. Fusions containing internal but not C-terminal regulatory domains manifested regulation of fusion protein level. Comparison of EPAS1 with HIF-1alpha demonstrated a similar organization for both proteins, and for the C terminus defined a conserved RLL motif critical for inducibility. Overall, EPAS1 sequences were less inducible than those of HIF-1alpha, and inducibility was strikingly reduced as their expression level was increased. Despite these quantitative differences, EPAS1 regulation appeared similar to HIF-1alpha, conforming to a model involving the modulation of both protein level and activity, through distinct internal and C-terminal domains.

Rejuvenation of degenerative thymus by oral melatonin administration and the antagonistic action of melatonin against hydroxyl radical-induced apoptosis of cultured thymocytes in mice.

The effect of melatonin on age-related thymic involution and apoptosis induced by hydroxyl radicals (*OH) in mouse thymocyte cultures was investigated. Exogenous melatonin was administered in the drinking water (15 microg/mL) of 7-month-old male Balb/c mice for 40 consecutive days. Our results show that melatonin distinctly reversed the age-related thymic involution as revealed by the notable increase of cellular density, particularly the number of thymocytes, percentage of thymocytes at G2+S phases and the younger morphological appearance as a whole when compared with control animals. More strikingly, the recovery of these morphometric parameters were maintained for 30 days after the termination of melatonin administration suggesting that the re-established homeostasis by melatonin may last for a longer time. At the same time, when primary culture of thymocytes was preincubated with 200 microM melatonin before their exposure to hydroxyl radicals (*OH) generated by Fe(2+)-mediated Fenton reaction, apoptotic cell death induced by *OH was almost completely prevented as determined by both flow cytometric analysis and the TUNEL assay. DNA laddering assay also documented the inhibition of thymocyte apoptosis by melatonin. Furthermore, we found that the *OH-induced increment of caspase-3 activity in thymocytes was completely abolished by melatonin preincubation. Taken together, our study indicates that in addition to other mechanisms, melatonin may also directly act as an antioxidant via attenuating apoptotic thymocyte death caused by free radicals and stimulates thymocyte proliferation in thymus and thus to rejuvenate the degenerative organ.

C. elegans EGL-9 and mammalian homologs define a family of dioxygenases that regulate HIF by prolyl hydroxylation.

HIF is a transcriptional complex that plays a central role in mammalian oxygen homeostasis. Recent studies have defined posttranslational modification by prolyl hydroxylation as a key regulatory event that targets HIF-alpha subunits for proteasomal destruction via the von Hippel-Lindau ubiquitylation complex. Here, we define a conserved HIF-VHL-prolyl hydroxylase pathway in C. elegans, and use a genetic approach to identify EGL-9 as a dioxygenase that regulates HIF by prolyl hydroxylation. In mammalian cells, we show that the HIF-prolyl hydroxylases are represented by a series of isoforms bearing a conserved 2-histidine-1-carboxylate iron coordination motif at the catalytic site. Direct modulation of recombinant enzyme activity by graded hypoxia, iron chelation, and cobaltous ions mirrors the characteristics of HIF induction in vivo, fulfilling requirements for these enzymes being oxygen sensors that regulate HIF.