[Revision rate of periprosthetic joint infection post total hip or knee arthroplasty of 34 hospitals in China between 2015 and 2017: a multi-center survey].

Objective: To investigate the rate of periprosthetic joint infection (PJI) revision surgeries and clinical information of hip-/knee- PJI cases nationwide from 2015 to 2017 in China. Methods: An epidemiological investigation. A self-designed questionnaire and convenience sampling were used to survey 41 regional joint replacement centers nationwide from November 2018 to December 2019 in China. The PJI was diagnosed according to the Musculoskeletal Infection Association criteria. Data of PJI patients were obtained by searching the inpatient database of each hospital. Questionnaire entries were extracted from the clinical records by specialist. Then the differences in rate of PJI revision surgery between hip- and knee- PJI revision cases were calculated and compared. Results: Total of 36 hospitals (87.8%) nationwide reported data on 99 791 hip and knee arthroplasties performed from 2015 to 2017, with 946 revisions due to PJI (0.96%). The overall hip-PJI revision rate was 0.99% (481/48 574), and it was 0.97% (135/13 963), 0.97% (153/15 730) and 1.07% (193/17 881) in of 2015, 2016, 2017, respectively. The overall knee-PJI revision rate was 0.91% (465/51 271), and it was 0.90% (131/14 650), 0.88% (155/17 693) and 0.94% (179/18 982) in 2015, 2016, 2017, respectively. Heilongjiang (2.2%, 40/1 805), Fujian (2.2%, 45/2 017), Jiangsu (2.1%, 85/3 899), Gansu (2.1%, 29/1 377), Chongqing (1.8%, 64/3 523) reported relatively high revision rates. Conclusions: The overall PJI revision rate in 34 hospitals nationwide from 2015 to 2017 is 0.96%. The hip-PJI revision rate is slightly higher than that in the knee-PJI. There are differences in revision rates among hospitals in different regions.

[Impacts of glycemic variability on the relationship between time in range and estimated glycated hemoglobin in patients with type 1 diabetes mellitus].

Objective: This study is to investigate the relationship between time in range (TIR) and glucose management indicator (GMI), and the impact of glycemic variability (GV) on their relationship in patients with type 1 diabetes mellitus (T1DM). Methods: The CGM data were collected from a multicenter randomized clinical trial of adults (≥18 years old) with T1DM, including 83 T1DM patients, respectively from the Third Affiliated Hospital of Sun Yat-sen University (72 cases), Drum Tower Hospital Affiliated to Nanjing University School of Medicine (2 cases), and the First Affiliated Hospital of University of Science and Technology of China (9 cases). All subjects wore the iProTM2 system for 14 days at baseline (0-2 weeks), 3 months (12-14 weeks), and 6 months (24-26 weeks). Data derived from iProTM2 sensor was used to calculate CGM parameters. Correlation between TIR and GMI was explored according to different stratification of glycemic variability assessed by glucose coefficient of variation (CV). Predicted TIR in the fixed GMI value was calculated via the linear regression equations performed in the respective interquartile group of CV. Results: From November 2017 to June 2021, a total of 233 CGM data were collected with 83 collected from baseline, 80 from the 3-month follow-up, 70 from the 6-month follow-up. Patients including 27 males had a median (Q1, Q3) age of 30.69 (25.22, 38.43) years, with a diabetes duration of 10.05(4.46, 13.92) years. The median (Q1, Q3) and effective wearing time of available CGM data was 13.92 (13.02, 14.00) days and 91.61% (84.96%, 95.94%), and the value of TIR, GMI and CV was 60.34%±13.03%, 7.14%±0.61% and 41.01%±7.64%, respectively. There was a strong negative correlation between TIR and GMI (r=-0.822, P<0.001). Multiple linear regression analysis showed that the predictive value of TIR calculated from a given GMI was 8.352% higher when CV was up to standard (36%) than that when CV was down to standard. Based on the multiple linear regression equations generated from quartiles of CV, the predicted TIR value was decreased across the ascending quartiles with 69.98 % in the lowest quartile of CV (≤35.91%), 64.57 % in 25th-50th quartile of CV (35.91%75th quartile, CV>45.86%) when GMI was set as 7%. Conclusions: There is a strong correlation between TIR and GMI in adult patients with T1DM in patients with type 1 diabetes mellitus. CV influenced the relationship between TIR and GMI.

Age-specific reference ranges of serum anti-müllerian hormone in healthy women and its application in diagnosis of polycystic ovary syndrome: a population study.

OBJECTIVE: To establish the age-specific centiles of serum anti-müllerian hormone (AMH) levels in Chinese women, and to explore the use of multiples of median (MoM) AMH levels for the diagnosis of polycystic ovary syndrome (PCOS). DESIGN: An observational study. SETTING: University-affiliated hospitals and community clinics. POPULATION: We included 3137 healthy women aged 20-44 years recruited prospectively or who had archived serum samples from previous research projects. Another validation cohort of 751 women with PCOS as well as ovulatory controls, which was a convenient sample of women attending for infertility or menstrual disorders, was also studied. METHODS: The serum samples were assayed for AMH by the automated Access AMH assay. MAIN OUTCOME MEASURES: Age-specific reference ranges were constructed on the primary cohort with the Lambda-Mu-Sigma method. The MoM AMH of each subject in the validation cohort was calculated. RESULTS: Centile curves of serum AMH level against age were established. MoM AMH was significantly higher in women with PCOS than in controls (P < 0.05). The area under the ROC curve was 0.852 (95% confidence interval [CI] 0.825-0.877) (P < 0.0001) for discriminating women with PCOS from ovulatory controls by MoM AMH. CONCLUSIONS: We established a set of year-by-year age-specific reference ranges of serum AMH levels in Chinese women. The MoM AMH derived from this set of reference ranges is a promising tool to replace antral follicle count in the diagnosis of PCOS. TWEETABLE ABSTRACT: A set of age-specific reference ranges of AMH levels was established in Chinese women. Multiples of median AMH may be used to diagnose PCOS.

[Impact of the COVID-19 epidemic on anxiety among the elderly in community].

Objective: To compare the prevalence of anxiety among old people before and during the COVID-19 epidemic in China, and to provide scientific evidence for psychological intervention of the elderly during public health emergencies. Methods: In 2019, the National Psychological Care Project for the Elderly was launched, covering 818 communities across the country, and 188 407 subjects received psychological assessment. In April and May 2020, a convenient sample of 6 467 aged 65 and above subjects were followed up on the anxiety status and its influencing factors during the epidemic period by using structurized questionnaire. Data collection and management were carried out using the national elderly psychological care project data collection platform. McNemar test was used to compare the difference of the prevalence of anxiety among elderly before (October 2019 to January 23, 2020) and during the epidemic (April-May 2020). The difference of the prevalence of anxiety among elderly with different characteristics was compared by chi square test. The influencing factors of anxiety before and during the epidemic situation were analyzed by multivariate logistic regression model. Results: The prevalence of anxiety symptoms in the elderly population was 4.95% (95%CI: 4.42%-5.48%) before the outbreak of COVID-19, and 10.10% (95%CI: 9.36%-10.83%) during the epidemic which was twice as high as before the outbreak. The difference was statistically significant (P<0.05). Multivariate logistic regression analysis showed that the risk factors of anxiety symptoms before the outbreak were with one underlying disease (OR=1.57, 95%CI: 1.05-2.37), with two or more underlying diseases (OR=3.10, 95%CI: 2.13-4.51), and the protective factors were with hobbies, good relationship between children, good relationship with spouse, positive aging attitude and good psychological resilience (all P<0.05). The risk factors of developing anxiety symptoms during the epidemic were living in rural areas (OR=1.77, 95%CI: 1.42-2.20), participating in social activities regularly (OR=1.23, 95%CI: 1.02-1.48), having a good relationship with friends (OR=1.42, 95%CI: 1.11-1.82) and were quarantined or people around were quarantined for medical observation (OR=2.80, 95% CI: 1.90-4.13). Conclusion: The COVID-19 epidemic leads to a double increase in anxiety among the elderly. We should pay more attention to the psychological state of the elderly in rural area and who is being quarantined or people around being quarantined for medical observation.

[Effect of exogenous gonadotropin dosage on embryo aneuploidy rate and pregnancy outcome in patients of preimplantation genetic test].

Objective: To investigate the effect of gonadotropin (Gn) on embryo aneuploidy rate and pregnancy outcome during preimplanptation genetic testing for aneuploidy (PGT-A) cycles. Methods: The clinical data of patients undergoing PGT-A cycle at the First Medical Center of the PLA General Hospital from January 1, 2013 to May 31, 2019 were retrospectively analyzed. Patients were divided into younger patient group (<35 years old) and elder patient group (≥35 years old) by maternal age, then divided into two groups in line with Gn dosage (≤2 250 U, >2 250 U), and into four groups by number of oocytes retrieved (1-5, 6-10, 11-15 and ≥16 oocytes). The embryo aneuploidy rate and pregnancy outcome between the groups were compared. Logistic regression was used to analyze the relationship between the cumulative amount of Gn, embryo aneuploidy rate and live-birth rate. Results: A total of 402 cycles (338 patients) and 1 883 embryos were included in the study. (1) In the younger patients, the aneuploidy rate was 52.5% (304/579) in the group of Gn≤2 250 U and 48.6% (188/387) in the group of Gn>2 250 U, with no significant difference between them (P=0.232). In the elderly patients, the difference in embryo aneuploidy rate between the two Gn group [57.9% (208/359) versus 60.6% (319/526)] was not statistically significant (P=0.420). (2) The embryonic aneuploidy rate in different protocol of ovary stimulation was analyzed,in the younger group, the embryonic aneuploidy rate in patients using antagonist long protocol was 50.3% (158/314), it was 50.0% (121/242) in agonist long protocol, 52.1% (207/397) in agonist short protocol and 6/13 in luteal phase protocol, no statistical difference was found in above groups (P=0.923); in the elder group, embryonic aneuploidy rate was 60.8% (191/314) in antagonist protocol, 58.4% (132/226) in agonist long protocol, 59.2%(199/336) in agonist short protocol, 5/9 in luteal phase protocol, respectively,no significant difference was found (P=0.938). (3) In the younger patients, the aneuploidy rate in 1-5 oocytes group, 6-10 oocytes group, 11-15 oocytes group and ≥16 oocytes group was 37.9% (11/29), 54.0% (94/174), 52.5% (104/198) and 50.1% (283/565) respectively, no significant difference was found between the groups (P=0.652); while in the elder patients, the difference between aneuploidy rate in each retrieved oocytes group [73.6% (89/121), 57.5% (119/207), 56.3% (108/192), 57.8% (211/365)] was statistically significant (P=0.046). (4) Logistic regression analysis of age, cumulative dosage of Gn, number of oocytes obtained, and embryo aneuploidy rate showed that there was no association between the amount of Gn and embryo aneuploidy rate (P>0.05); the increase in maternal age would increase the risk of aneuploidy rate of embryos, which was statistically significant (OR=1.031, 95%CI: 1.010-1.054, P=0.004); the increase in oocytes retrived would significantly decrease the risk of aneuploidy (OR=0.981, 95%CI: 0.971-0.991, P<0.01). (5) There was no significant difference in biochemical pregnancy rate [55.6% (80/144) versus 52.1% (63/121)], clinical pregnancy rate [50.0% (72/144) versus 47.9% (58/121)] and live-birth rate [46.5% (67/144) versus 40.5% (49/121)] between different Gn dosage groups (P=0.613, P=0.738, P=0.324). The logistic regression analysis showed that the maternal age, the cumulative dosage of Gn, the number of oocytes obtained, and the ovarian stimulation protocol had no effect on the live-birth rate (all P>0.05). Conclusions: In PGT-A cycle, the dosage of Gn has no association with the embryo aneuploidy rate and pregnancy outcome. In the patients ≥35 years old, the increase in number of oocytes obtained may decrease the risk of aneuploidy. Age is an important factor affecting the embryo aneuploidy in PGT-A cycle.

[Microbiology analysis of periprothetic joint infection post total hip and knee arthroplasty of 9 centers in Beijing between 2014 and 2016].

Objective: To investigate the microbiological test, antibiotic sensitivity and surgical treatment of periprosthetic joint infection(PJI) cases in post total hip arthroplasty (THA) and total knee arthroplasty (TKA) patients. Methods: A retrospective cross-sectional survey was conducted on 318 patients who underwent THA or TKA in 9 clinical centers in Beijing from January 2014 to December 2016.The data of microbiology, antibiotic sensitivity and surgical treatment were collected.The average age of patients was (62.3±13.1) years old (range: 21-86 years old), including 145 males and 173 females.The body mass index was (25.6±3.8) kg/m (2) (range: 15.6-38.1 kg/m(2)). Results: In total, 318 patients had microorganisms detected by periprosthetic tissue culture or synovial fluid culture, 209 cases (65.7%) had Gram-positive bacteria, 29 cases (9.1%) had Gram-negative bacteria, 10 cases (3.1%) had fungi, 3 cases (0.9%) had non-tuberculous mycobacteria, 72 cases (22.6%) were negative, 69 cases (21.7%) had methicillin-resistant bacteria. The antibiotic sensitivity results showed that the overall resistance rate of penicillin, cefuroxime, amoxicillin+clavulanic acid was 79.9%, 69.9%, and 68.1%, respectively; meropenem, vancomycin, and linezolid resistance rate was 0. For the treatment methods of hip and knee PJI, two-stage revision surgery acounted for 72.9% (108/148) and 64.1% (109/170), respectively. One-stage revision surgery accounted for 21.6% (32/148) and 7.6% (13/170), and open debridement surgery accounted for 4.7%(7/148) and 26.4% (45/170). Conclusions: Gram-positive bacteria was still the main pathogen of PJI.The methicillin-resistant bacteria and rare bacteria should be payed attention to. The Majority of hip and knee PJI cases were treated by two-stage revision surgery.

Layering Poly (lactic-co-glycolic acid)-based electrospun membranes and co-culture cell sheets for engineering temporomandibular joint disc.

The temporomandibular joint disk (TMJD) lacks blood vessels and is characterized by slow self-repair. Qualitative lesions in TMJD are difficult to repair. In this study, electrospun poly (lactic-co-glycolic acid) (PLGA) scaffolds were used to reconstruct temporomandibular joint discs by tissue engineering. Rabbit temporomandibular joint disc cells (TMJDCs) and rabbit synovium-derived mesenchymal stem cells (SMSCs) were co-cultured in 1:1 ratios. Cell sheets were induced by ascorbic acid incubated with electrospun PLGA scaffolds for 14 days in the presence (10 ng/ml in culture medium) or absence of TGF-ß3. Dimethylmethylene Blue Assay (DMMB) was used to determine the content of glycosaminoglycans in the extracellular matrix. The expression of Col1a1, Col2a1, Sox-9 and Runx-2 was quantified by RT-PCR, and the expression of type II collagen was observed by immunofluorescent staining. After 14 days of cultivation, the electrospun PLGA scaffold-loaded cell sheets could form an articular disc tissue with certain morphological characteristics. The expression of chondrogenic-related genes (Col2a1, Sox-9) and the secretion of extracellular matrix (GAG, type II collagen) in the co-culture group were close to those in the TMJDC group alone. The results suggest that PLGA electrospun scaffold-loaded co-cultured cell membrane could be used in the tissue engineering reconstruction of the temporomandibular joint disc.

Effectiveness of preoperative decolonization with nasal povidone iodine in Chinese patients undergoing elective orthopedic surgery: a prospective cross-sectional study.

Staphylococcus aureus colonization in the nares of patients undergoing elective orthopedic surgery increases the potential risk of surgical site infections. Methicillin-resistant S. aureus (MRSA) has gained recognition as a pathogen that is no longer only just a hospital-acquired pathogen. Patients positive for MRSA are associated with higher rates of morbidity and mortality following infection. MRSA is commonly found in the nares, and methicillin-sensitive S. aureus (MSSA) is even more prevalent. Recently, studies have determined that screening for this pathogen prior to surgery and diminishing staphylococcal infections at the surgical site will dramatically reduce surgical site infections. A nasal mupirocin treatment is shown to significantly reduce the colonization of the pathogen. However, this treatment is expensive and is currently not available in China. Thus, in this study, we first sought to determine the prevalence of MSSA/MSRA in patients undergoing elective orthopedic surgery in northern China, and then, we treated the positive patients with a nasal povidone-iodine swab. Here, we demonstrate a successful reduction in the colonization of S. aureus. We propose that this treatment could serve as a cost-effective means of eradicating this pathogen in patients undergoing elective orthopedic surgery, which might reduce the rate of surgical site infections.

Is intralesional resection suitable for central grade 1 chondrosarcoma: A systematic review and updated meta-analysis.

BACKGROUND: The surgical choice for grade 1 chondrosarcoma has been debated for decades. Intralesional resection can minimize the damage caused by surgery and offer better functional outcome. However, controversy remains about whether it will result in higher rates of local recurrence and metastasis, fewer complications, and better functional outcome compared with resection with wide margin. This systematic review and updated meta-analysis therefore compared intralesional resection and resection with wide margin in terms of local recurrence, metastasis, complications, and functional outcome. METHODS: Medline, Embase, and the Cochrane Library were comprehensively searched in December 2016 to identify studies comparing intralesional resection and resection with wide margin for central grade 1 chondrosarcoma. Data of interest were extracted and analyzed using Review Manager 5.3. RESULTS: Ten studies involving 394 patients were included, with 214 patients who had intralesional resection and 180 patients who had resection with wide margin for grade 1 chondrosarcoma. Intralesional resection was associated with lower complication rates (P < 0.0001) and better Musculoskeletal Tumor Society score (MSTS). There were no significant differences in terms of overall local recurrence (P = 0.27), local recurrence based on adjuvant therapies (P = 0.22), local recurrence in studies that included lesions of the hand, foot, pelvis, and axial skeleton (P = 0.55), and metastasis (P = 0.74) between groups. CONCLUSION: Intralesional resection provides lower complications and better functional outcome with no significant increase in the risk of recurrence and metastasis. We think it is a suitable treatment for central grade 1 chondrosarcoma.

Bovine antibody dependent cell-mediated cytotoxicity (ADCC) effector cells.

ADCC effector cells from bovine blood were separated by centrifugation, adherence and rosetting techniques. Each enriched cell population, peripheral blood mononuclear cells (PBM), null lymphocytes, monocytes and neutrophils, was then examined for its capacity to mediate ADCC. Utilizing heterologous sensitizing antisera it was found that monocytes had approximately twice the ADCC activity of null lymphocytes and that neutrophils had essentially no activity. However, when homologous sensitizing antisera were used it was found that neutrophils possessed the greatest activity followed by monocytes and null cells. Results confirm the existence of an ADCC active null lymphocyte in the bovine.

[Determination of docosahexenoic acid in human serum by capillary gas chromatography].

A reliable CGC method was developed for the determination of docosahexenoic acid (DHA) in human serum. The serum sample was acidified by adding two drops of 4.5 mol.L-1 H2SO4, and DHA was extracted from serum using ethyl acetate. The extract was evaporated to dryness under nitrogen stream. To each residue, 1 ml 1.3 mol.L-1 methanolic hydrogen chloride solution was added and the mixture was allowed to stand for 30 min in 60 degrees C water bath. After derivatization, the mixture was extracted with 1 ml of n-hexane. The solvent was evaporated under a stream of nitrogen to dryness and the residue was dissolved in 30 microliters n-hexane and subjected to capillary GC, which was equipped with a fused silica capillary column (26.3 m x 0.25 mm ID) coated with FFAP (free fatty acid phase, 0.1 micron film thickness). Tricosanoic acid was used as an internal standard. The retention time of DHA-M and internal standard was 23.41 min and 20.79 min, respectively. The minimum detection concentration of DHA in serum was 40 ng.ml-1 with a serum volume of 200 microliters and S/N value of 2. A good linear relationship between the peak area ratios and concentrations was found at the DHA concentrations ranging from 25 to 200 micrograms.ml-1. The within-day and between-day precision was 5% and 9%, respectively.

Effectiveness of preoperative decolonization with nasal povidone iodine in Chinese patients undergoing elective orthopedic surgery: a prospective cross-sectional study

Staphylococcus aureus colonization in the nares of patients undergoing elective orthopedic surgery increases the potential risk of surgical site infections. Methicillin-resistant S. aureus (MRSA) has gained recognition as a pathogen that is no longer only just a hospital-acquired pathogen. Patients positive for MRSA are associated with higher rates of morbidity and mortality following infection. MRSA is commonly found in the nares, and methicillin-sensitive S. aureus (MSSA) is even more prevalent. Recently, studies have determined that screening for this pathogen prior to surgery and diminishing staphylococcal infections at the surgical site will dramatically reduce surgical site infections. A nasal mupirocin treatment is shown to significantly reduce the colonization of the pathogen. However, this treatment is expensive and is currently not available in China. Thus, in this study, we first sought to determine the prevalence of MSSA/MSRA in patients undergoing elective orthopedic surgery in northern China, and then, we treated the positive patients with a nasal povidone-iodine swab. Here, we demonstrate a successful reduction in the colonization of S. aureus. We propose that this treatment could serve as a cost-effective means of eradicating this pathogen in patients undergoing elective orthopedic surgery, which might reduce the rate of surgical site infections.

Cryoloop vitrification of rabbit oocytes.

BACKGROUND: Vitrification is assumed to be a promising method to cryopreserve human oocytes but still needs optimization. In this study, rabbit oocytes (fertilized by ICSI) were vitrified with cryoloops, and the effect of three different cryopreservation protocols on spindle configuration and embryo quality was assessed. METHODS: Metaphase II rabbit oocytes were randomly assigned to one of four groups: (i) control; (ii) E40 [40% ethylene glycol (EG)]; (iii) ED20 [20% EG + 20% dimethylsulphoxide (DMSO)]; and (iv) ED20 + M (20% EG + 20% DMSO + vitrification machine). After warming, one part of each group was fertilized by ICSI to examine the fertilization and embryo cleavage ability, and the others were immunostained for tubulin and chromatin before visualization using confocal microscopy. RESULTS: The survival rates after warming were 79.1, 83.1 and 82.3%, respectively. In protocols E40 and ED20, the spindles were severely injured and the embryo quality not good compared with those in the ED20 + M group. CONCLUSIONS: The fastest cooling rate in combination with EG and DMSO as cryoprotectants had the fewest adverse effects on the spindle configuration of rabbit oocytes and embryo development.

Promoter function and the role of cytokines in the transcriptional regulation of rabbit CYP2E1 and CYP2E2.

Rabbit CYP2E1 and CYP2E2 show considerable similarity in the 5' flanking region, but a 32-base-pair element (32-BPE) that is repeated in 2E1 is present only as a single inexact copy in 2E2. In the present investigation, footprinting disclosed two specific binding sites for liver nuclear proteins, and the DNase I sensitivity profiles of the two genes were found to be different. Several positive and negative regulatory elements were identified by transfection with a series of constructs of upstream CYP2E sequences fused to the luciferase gene. Both genes have an HNF-1 consensus motif with one nucleotide mismatch, which affects binding affinity and promoter activity. Investigation of DNA-protein interactions revealed that Sp1 and NFkappaB bind exclusively to the 32-BPE of 2E1 and 2E2, respectively, suggesting a possible regulatory role for the 32-BPE. Interleukin-1alpha (IL-1alpha) gave rise to a 2.5-fold increase in the promoter activity of 2E1 in HepG2 cells, and the IL-1alpha-mediated induction of reporter gene expression was almost completely prevented when the 32-BPE was deleted. Increased DNA binding and Sp1 protein content as a result of IL-1alpha treatment, as well as cotransfection experiments with pPacSp1, suggest that Sp1 is a transcription activator for the induction of 2E1 by IL-1alpha in HepG2 cells.

Regulation of rabbit cytochrome P450 2E1 expression in HepG2 cells by insulin and thyroid hormone.

The regulation of cytochrome P450 (CYP) 2E1, the ethanol-inducible isoform, is particularly complex. The level is affected by a variety of other foreign compounds, by insulin (as studied in several laboratories), and by triiodothyronine (T3), which has not been previously examined at the molecular level. In the present investigation, a stably transfected HepG2 cell line harboring a rabbit CYP2E1 minigene containing the coding sequence together with 1.6 kilobases of the 5' flanking region and the untranslated region (UTR), as well as 0.5 kilobases of the 3' UTR, was established. Western blot analysis showed that 1 microM insulin decreased the CYP2E1 protein level in a dose- and time-dependent manner, whereas 1 microM T3 increased the level 2-fold in 1 day and 8-fold in 5 days. Similarly, steady state CYP2E1 mRNA levels were decreased by insulin but were increased by T3. Neither hormone affected the transcription rate of the CYP2E1 5' flanking region with an UTR/luciferase fusion gene, indicating that the regulation is post-transcriptional in this system under our experimental conditions. When the CYP2E1 3' UTR was removed from the minigene, CYP2E1 mRNA and protein were up-regulated by insulin but were not affected by T3. These findings, including mRNA half-life determinations, indicate that the effects of insulin and T3 are a result of altered mRNA stability and that the 3' UTR of CYP2E1 contains regulatory information for these hormone-mediated effects.

Structure-function analysis of CYP2A10 and CYP2A11, P450 cytochromes that differ in only eight amino acids but have strikingly different activities toward testosterone and coumarin.

Cytochrome P450 NMa, which was first identified in this laboratory in rabbit nasal microsomes, is now known to represent two distinct gene products, P450s 2A10 and 2A11. In the present study, chimeric and site-directed mutants of 2A11 were constructed to determine which of the eight different amino acid residues are responsible for the much greater activity of 2A10 toward coumarin and testosterone. Mutation of Arg62 and Asp63 of 2A11 to the corresponding residues in 2A10, or mutation of Thr120 to Ser, as found in 2A10, did not change the activities. However, mutation of Arg62, Asp63, Gln104, Ala117, and Thr120 of 2A11 to the corresponding residues in 2A10 resulted in a protein that is as active as 2A10 in coumarin hydroxylation and approximately half as active as 2A10 in androstenedione formation. Mutation of Arg372 in 2A11 to His, as found in 2A10, resulted in a significant increase in the rate of hydroxylation of testosterone, but not of coumarin. Our findings indicate that the identify of the amino acid at position 104 and/or 117 is important for activity with testosterone and for regioselectivity at the 17 position, as well as for optimal activity with coumarin. In contrast, the identity of the residue at position 372 is important for optimal activity with testosterone but not the regioselectivity at the 17 position and does not influence the activity with coumarin.

Isolation and heterologous expression of cloned cDNAs for two rabbit nasal microsomal proteins, CYP2A10 and CYP2A11, that are related to nasal microsomal cytochrome P450 form a.

Nasal microsomal P450 form a (NMa), a major cytochrome P450 isozyme in rabbit olfactory and respiratory nasal mucosa with high activity toward a variety of odorants and environmental toxicants, was previously purified to electrophoretic homogeneity from rabbit nasal microsomes. In the present study, a cDNA library constructed from poly(A)+ RNA from rabbit respiratory nasal mucosa was screened with antibodies to P450 NMa, and five immunopositive clones were isolated and characterized. Sequence analysis indicated that the clones encode two highly similar P450s that contain 494 amino acid residues, with the first 20 corresponding to P450 NMa, and differ from each other in only 8 residues scattered throughout the polypeptide chains. On the basis of structural homology the two proteins are designated as CYP2A10 and CYP2A11 and are the first members of the P450 2A subfamily to be identified in nasal tissue. Genomic blot analysis indicated that 2A10 and 2A11 are apparently not allelic variants. Both genes are expressed in liver and lung as well as in nasal tissues, as judged by RNA blot analysis, but the relative levels of the two mRNAs differ. Both enzymes were partially purified after expression of the cDNAs in Escherichia coli and shown to catalyze the oxygenation of a variety of substrates, including ethanol and procarcinogens such as N-nitrosodiethylamine and phenacetin. P450 2A10 is generally more active than P450 2A11 and strikingly so in the conversion of testosterone to androstenedione.

Cytochromes P450 NMa, NMb (2G1), and LM4 (1A2) are differentially expressed during development in rabbit olfactory mucosa and liver.

Mammalian olfactory mucosa has a high concentration of cytochrome P450 monooxygenases (P450). The major olfactory P450 isoforms in adult rabbits include P450 NMa, which is found in both olfactory and respiratory mucosa, as well as in liver at a low level, P450 NMb (2G1), which is olfactory specific, and P450 form 4 (1A2), which is found only in liver and olfactory mucosa. In the present study, we have found that the developmental expression of olfactory P450 in rabbits is not coordinated with the ontogenesis of hepatic P450. These three P450 isoforms were detected immunochemically and found to be at a relatively high level in olfactory but not hepatic microsomes in the first 2 weeks after birth. In the liver, NMb is not detectable at any age and NMa is not detectable until the fourth week. P450 1A2 is not detectable until the third week, but its level increases rapidly in the fourth week. These P450 isoforms are also detectable in prenatal olfactory tissue at 2 days before birth, indicating that direct exposure to air is not a prerequisite for their early expression in this tissue and that the early appearance of these enzymes may be controlled by both endogenous and environmental factors. In addition, the developmental expression of 2E1, a minor olfactory P450 isoform, also occurs earlier in olfactory mucosa than in liver, and the same conclusion can be made about the expression of NADPH-P450 reductase, which is detectable in olfactory microsomes but not in hepatic microsomes from prenatal rabbits. Thus, the regulatory mechanisms that control basal prenatal expression in the olfactory tissue may be common for multiple P450 isoforms and perhaps also for other biotransformation enzymes. The tissue-specific early onset of expression of multiple forms of P450 in olfactory tissue suggests that these enzymes may play an important role in the neonatal period, when olfactory ability is vital for the survival of the newborn. The presence of relatively high levels of biotransformation enzymes in the olfactory mucosa may also have important implications for neonatal inhalation toxicology.