[Long-term evaluation and physical and mental effects of residual tinnitus following treatment of sudden hearing loss].

Objective: To clarify the long-term characteristics of tinnitus following treatment of sudden deafness and its long-term physical and mental effects on patients. Methods: A retrospective analysis was performed on 88 patients (46 males and 42 females; Age from 11 to 89 years) with sudden deafness treated in Department of Otoscope Surgery of Peoples's Libration Army General Hospital in Beijing from April 2020 to January 2021, and the occurrence of tinnitus and treatment effect of all patients were analyzed. Follow-up was conducted for patients with residual tinnitus after treatment for more than 1 year by the investigation and filling in the survey information collection form, Tinnitus Evaluation Questionnaire (TEQ) and Tinnitus Handicap Inventory (THI). Descriptive statistics and SPSS 22.0 software were used for statistical data analysis. Results: In this study, 93.2% (82/88) of patients with sudden deafness were accompanied by tinnitus at the onset, and the proportion of long-term tinnitus after treatment was 90.2% (74/82). After 1 year of treatment for sudden deafness, the improvement of tinnitus was significant in low-frequency sudden deafness compared with those of high-frequency, flat and total deafness sudden deafness (χ2 value was 6.801, 4.568 and 4.038, all P<0.05). In patients with residual tinnitus, 9 (12.2%) patients felt minimal loudness or even no loudness, 34 (46.0%) patients felt slight loudness, 28 (37.8%) patients felt tinnitus was relatively loud, and 3 (4.1%) patients felt tinnitus was loud or noisy. Nine (12.2%) patients's sleep was often affected, 41 (55.4%) patients's sleep was sometimes affected, 9 (12.2%) patients's sleep was rarely affected, 15 (20.3%) patients's sleep was almost not affected. Twenty-eight (37.8%) patients basically completely adapted to tinnitus and 46 (62.2%) patients did not completely adapted to residual tinnitus. Eight (10.8%) patients had no impact on life, 39 (52.7%) patients had slight impact, 22 (29.7%) patients had moderate impact, and the other 5 (6.8%) patients had greater impact. According to tinnitus evaluation questionnaire(TEQ), there were 12 cases (16.2%) of grade Ⅰ, 26 cases (35.1%) of grade Ⅱ, 28 cases (37.8%) of grade Ⅲ, 7 cases (9.5%) of grade Ⅳ and 1 case (1.4%) of grade Ⅴ. According to tinnitus handicap inventory(THI), tinnitus disability was classified into grade Ⅰ, 22 cases (29.7%), grade Ⅱ, 14 cases (18.9%), Grade Ⅲ, 27 cases (36.5%) and grade Ⅳ, 11 cases (14.9%). Conclusion: The rate of residual tinnitus following treatment of sudden deafness is high. Some of the patients can completely adapt residual tinnitus after one year, but some of them will be affected when sleep, work and study. Residual tinnitus can lead to tinnitus disability in different degrees.

The effect of cold exposure on the levels of glucocorticoids, 11-hydroxysteroid dehydrogenase 2, and placental vascularization in a rat model.

OBJECTIVE: Cold exposure (CE) before birth is one of the initial stressors that may impact mammalian pregnancy, changing placental and fetal development and affecting the health of the offspring. While glucocorticoids (GCs) participate in the body's response to the stress of CE, the specific mechanisms of their action are unclear. This study aims to determine the effect of CE stress on the placenta and to test whether stress, caused by cold exposure in pregnancy impairs fetal development by changing placental angiogenesis via excessive GC expression. MATERIALS AND METHODS: CE rat model was created by exposing 30 SD rats to cold preconception, or during the first, second, and third weeks of pregnancy. Serum cortisol and soluble fms-like tyrosine kinase-1 (sFlt-1) expression levels, physiological index changes (food intake, body weight change and blood pressure), and pregnancy outcomes (fetal rat weight, number of live fetal rats, and placental weight) were collected at baseline and at different time points after the conception. Protein expression levels of 11 ß-hydroxysteroid dehydrogenase 2 (11ß-HSD2), glucocorticoid receptor, vascular endothelial growth factor A (VEGF-A), placental growth factor (PIGF), and sFlt-1 in placental tissues were measured by western blotting. Cytokeratin (CK) and laminin (LN) in trophoblasts, and α-actin in vascular smooth muscle of the spiral arteries of pregnant rats after the systemic cold treatment were assessed by immunofluorescence and visualized by fluorescent microscopy. To test the effect of 11ß-HSD2 levels on the placental recasting, human first-trimester extravillous trophoblast cells (HTR8/SVneo) underwent knockdown using specific 11ß-HSD2 siRNA constructs.  Expression levels of 11ß-HSD2 were analyzed by quantitative real-time PCR (qPCR) and into HTR8 cells, and the expression levels of the 11ß-HSD2 gene in each group were measured using qPCR. Cell migration and invasion was assessed by Transwell migration assay, and sFlt-1 levels in HTR8 cells were measured by ELISA. RESULTS: CE pre-conception led to consistently increasing serum corticosterone and sFlt-1 levels throughout pregnancy, and persistently increased diastolic blood pressure (DBP) in rat CE model compared to control animals. CE during the second week of gestation (Gp.3) was associated with significantly lower placental weight (p=0.0003). Cold exposure in the third week (Gp.4) was associated with significantly (p=0.001) lower fetal weight. CE pre-conception was associated with significantly decreased placental levels of 11ß-HSD2, glucocorticoid receptor, VEGF-A, PIGF, and sFlt-1 proteins and α-actin compared to the control group. Silencing 11ß-HSD2 by siRNA led to reduced cell migrations and invasion, and markedly increased expression levels of sFlt-1 in HTR8/SVneo cells (p<0.05). CONCLUSIONS: Pre-conception cold exposure and during early pregnancy leads to increased GCs levels and impaired placental 11ß-HSD2 activity. We suggest that the subsequent 11ß-HSD2-induced increase in the sFlt-1expression during early pregnancy may affect placental vascular remodeling and change placental morphological structure and function.

[The prevalence and risk factors of diabetic peripheral artery disease in Chinese communities].

Objective: To investigate the prevalence and risk factors of diabetic peripheral artery disease (PAD) in patients with type 2 diabetes mellitus (T2DM) managed in primary health care in China. Methods: A total of 2 528 T2DM patients were selected using a two-stage cluster random sampling method based on the baseline survey of the "China Diabetic Foot Prevention Model Project." The study was conducted in 2015 among T2DM patients in 8 primary healthcare centers in Changshu county and Jiang'an district of Wuhan, China. Data collection methods included a questionnaire, body measurement, and blood glucose detection. The Ankle-Brachial Index (ABI) is the most widely used noninvasive vascular test. A binary logistic regression model was used to analyze the influence factors. Results: The prevalence of PAD was 11.2% among the diabetic patients managed in primary health care in the two cities. The prevalence of PAD under 55 years old, 55- years old, 65- years old, and ≥75 years old were 7.8%, 6.0%, 12.9% and 22.5%, respectively. Multivariate stepwise logistic regression identified influence factors included older age, higher education level, smoking, drinking, postprandial glucose uncontrol, and prior myocardial infarction or angina. Compared to age <55 years, the odds ratio for PAD were 0.74 for 55- years (95%CI: 0.43-1.28), 1.72 for 65- years (95%CI: 1.05-2.81), 3.56 for 75 years and above (95%CI: 2.07-6.11), respectively. Compared to patients with education in primary school and below, the odds ratio was 1.37 (95%CI: 0.97-1.94), 2.48 (95%CI: 1.73-3.55), 1.99 (95%CI: 1.26-3.13) for those with education levels of junior high school, senior high school, and college, respectively. Current smoking (OR=1.49, 95%CI: 1.02-2.17), current drinking (OR=0.45, 95%CI: 0.28-0.71), postprandial glucose uncontrol (2 h postprandial plasma glucose >10.0 mmol/L: OR=1.72, 95%CI: 1.22-2.43), and prior myocardial infarction or angina (OR=2.32, 95%CI: 1.50-3.61) were influencing factors of PAD. Conclusions: Despite the high prevalence of PAD in diabetes managed in primary health care; multiple risk factors are not effectively aware of and under control. It is urgent to promote ABI screening and standardized management for diabetes, especially in primary health care.

[Analysis of clinical characteristics of middle ear osteoma at different locations].

Objective: To explore the clinical characteristics, intervention and treatment of tympanic osteoma at different locations. Methods: The medical history, audiological and imaging examination, operation and follow-up results of two patients with tympanic osteoma at different sites were reviewed and summarized. Furthermore, the clinical characteristics and interventions of 36 patients reported in literatures with tympanic osteomas were also summarized and analyzed. Results: Osteoma of the two patients collected in this study located at promontory and incus respectively;both of them presented with intact tympanum and conductive deafness, without obvious etiology or predisposing factor. Both of them underwent surgeries and the hearing improved significantly. For patient one, the ossicular chain was intact and restored to activity after removed the osteoma. For patient two, an artificial ossicle was implanted after removed the osteoma and incus. In the 36 patients reported in literatures, the average age was 26.5 years, and 39.47% of them located at promontory; in addition, the main symptoms of them were progressive hearing loss, tinnitus and ear stuffy. Conclusions: Patients with tympanic osteoma are characterized by conduction deafness with intact tympanic membrane, and the most common lesion is promontory. Hearing can be restored by excision of the osteoma and maintenance or reconstruction of the ossicle chain.

[Identification of a novel mutation of SOX10 gene and analysis of the phenotype].

Objective: To explore the clinical features and pathogenic mechanisms of a special syndrome with congenital sensorineural hearing loss, albinism, heterochromia iridis, nystagmus and myelin dysplasia. Methods: Detailed medical history, systematic audiology tests, ophthalmic and neurological examinations were carried out to analyze the clinical features of the child, and further molecular genetic tests including chromosome karyotype analysis, and deafness gene screening were conducted. Results: A new de novo heterozygous mutation (c.336G>T/p.Met112Ile) was detected in the child, while both his parents were demonstrated to be wild-type and symptom free. The analysis of clinical features indicated the diagnosis of PCW syndrome. Conclusion: This study identified a new mutation of SOX10 gene, which enriched the mutation spectrum of this gene. And the analysis of clinical characteristics of this patient also expanded the phenotype of this gene. This study provided a reference for clinical diagnosis and genetic diagnosis of PCW syndrome.

[Association between low handgrip strength and air pollution among people aged 50 years and over].

Objective: To examine the association between long-term exposure to ambient PM(2.5) combined with indoor air pollution and handgrip strength among people aged 50 and over. Methods: Data were from the first wave of World Health Organization Study on global AGEing and adult health in China. Ambient annual concentration of PM(2.5) was estimated by using the satellite data we also investigated the use of fuels and chimneys as indoor air pollution. A two-level (individual level and community level) linear model was applied to examine the association between long-term exposure to ambient PM(2.5) combined with indoor air pollution and the handgrip strength. Results: A total of 13 175 individuals aged 50 years and over were included for analysis. The handgrip strength was (26.67±0.54) kg. Ambient PM(2.5) was found to be significantly associated with the risk of decreased handgrip strength. Outdoor PM(2.5) concentration was negatively correlated with handgrip strength (ß=-0.23, 95%CI: -0.31 - -0.14) decrease in handgrip strength after adjusting for gender, age, residence, education, household assets, intake of vegetables and fruits, smoking and drinking, physical activity. In rural area, compared to those who used solid fuel, use of clean fuel increased (ß=1.41, 95%CI: 0.36-2.46) handgrip strength. But in urban area, we did not find any statistically significant association between the use of clean fuel and handgrip strength (ß=0.19, 95%CI: -0.95-1.32). Conclusion: This study found that long-term exposure to ambient PM(2.5) combined with indoor air pollution was significantly associated with low handgrip strength among people aged 50 years and over, this suggested that ambient PM(2.5) might serve as one of the risk factors for low physical function seen in the people aged 50 years and over.

[Mitochondrial DNA deletion syndrome: a case report and literature review].

Summary Mitochondrial DNA（mtDNA） deletion is a rare occurrence that results in defects to oxidative phosphorylation. The common clinical presentations of mtDNA deletion vary but include mitochondrial myopathy, Pearson syndrome, Kearns-Sayre syndrome, and progressive external ophthalmoplegia. However, in clinical practice, some cases cannot be classified as any typical syndrome due to the absence or overlap of phenotypes. Here, we report one case of a 5-year-old girl who presented with progressive deterioration of her clinical status, which included systemic electrolyte disturbance, Fanconi syndrome and sensorineural hearing loss. Through a combination of systematic examinations and molecular analyses, mitochondrial disease was confirmed. A novel 6991-base pair deletion（deletion of mtDNA nt 7808-14798） was identified which confirmed molecular pathogeny of patient. Following treatment, the patient was stabilized and her hearing loss improved by hearing aid. This paper provided an important reference for the diagnosis and treatment of similar patients in clinical practice.

[Case report of familial microtia combined with tetralogy of Fallot and scoliosis and literature review].

Summary Microtia is a kind of malformation affecting the development of the external ear and middle ear. In China, researches have pointed out that the incidence of microtia was 3.06 per 10 000 people. About 40% of patients with microtia were identified with other systemic malformation, and the commom complications included congenital heart disease, scoliosis, anophthalmia, cleft palate, facial asymmetry, facial asymmetry, etc. Of which, the prevalence of microtia with congenital heart disease was 18.5%, and it was 7% of patients with scoliosis. It is very rare for patients of microtia combined with multi-malformations. In this study, we reported a case of familial microtia combined with tetralogy of Fallot and scoliosis, and undertook a systematic review of the literature.

Chronic obstructive pulmonary disease deaths, disability-adjusted life years, and risk factors in Hubei province of mid-China, 1990-2015: the Global Burden of Disease Study 2015.

OBJECTIVES: The trends of chronic obstructive pulmonary disease (COPD) mortality and prevalence from 1990 to 2015 in Hubei province of mid-China remain unknown. We used findings from the Global Burden of Disease (GBD) 2015 study to estimate the COPD burden and attributable to risk factors in Hubei province of China from 1990 to 2015. STUDY DESIGN: The GBD study uses various analytical tools and a diverse set of data sources to generate comparable estimates of deaths and mortality rates broken down by age, sex, cause, year, and geography. METHODS: Data were extracted from the GBD 2015 study. Statistical models were used to produce comprehensive results of COPD deaths, prevalence, disability-adjusted life years (DALYs), years of life lost, years lived with a disability, and attributable to risk factors in Hubei. The median of the percent change and 95% uncertainty intervals were determined for the period between 1990 and 2015. RESULTS: In 2015, there were 37,144 deaths from COPD in Hubei, accounting for 10.05% of the total deaths in Hubei. The age-standardized COPD death rate was reduced by 60.28% from 188.67 per 100,000 (in 1990) to 74.94 per 100,000 (in 2015). The age-standardized prevalence decreased from 4.30% (1990) to 2.85% (2015). By 2015, the leading risk factors for all ages COPD DALYs were smoking and ambient particulate matter pollution, accounting for 44.69% and 32.91%, respectively. The proportion of ambient ozone pollution was increasing steadily each year since 1990. CONCLUSION: Hubei has made substantial progress in reducing the mortality due to COPD since 1990, but the absolute number of COPD cases is increasing steadily, given the population growth and aging. The increasing contribution from smoking, ambient particulate matter pollution, and ambient ozone pollution should require growing attention.

[The effects of postauricular injection of methylprednisolone on medium-high frequency sudden hearing loss].

Objective:To study the effects of postauricular injection of methylprednisolone on medium-high frequency sudden hearing loss.Method:The data of 45 patients with invalid treatment who were diagnosed as medium-high frequency sudden hearing loss were retrospectively studied. They were divided into postauricular injection group and non-postauricular injection group. The treatment efficiency and hearing improvement at each frequency were compared between the two groups.Result:The improvements of hearing threshold in the non-postauricular injection group were(6.30±5.00)dB HL,(3.80±5.52)dB HL,(5.35±5.50)dB HL,(15.60±11.84)dB HL,(19.60±13.46)dB HL,(15.40±12.90)dB HL at 250, 500, 1000, 2000, 4000 and 8000Hz, respectively. The hearing improvements of the postauricular injection group were (5.35±6.22)dB HL,(2.50±3.00)dB HL,(4.65±6.60)dB HL,(23.75±10.75)dB HL,(25.75±11.73)dB HL,(30.50±14.50)dB HL at 250, 500,1000,2000,4000 and 8000Hz, respectively. There were significant differences between the two groups in hearing improvements at 2000-8000Hz. The treatment effective rates were 44% and 80% for the non-postauricular injection group and postauricular injection group respectively, which showed a significant difference(χ²=8.385P<0.05).Conclusion:Postauricular injection of methylprednisolone as a remedy treatment is safe and effective for sudden hearing loss in middle and high frequency.

[Investigation of CXCR4 mediated chemoresistance in nasopharyngeal carcinoma cell line CNE2].

Objective:Since nasopharyngeal carcinoma is easy to develop resistance during cisplatin-based chemotherapy,CXCR4 expression levels were elevated in mang tumors,and the factor to do with tumor metastasis and chemotherapy drug resistance,and so on has a very important link.We established cisplatin-resistant nasopharyngeal carcinoma cell line, named as CNE2/DDP, and investigated the function of CXCR4 in molecular mechanism behind this resistance.Method:CNE2/DDP was firstly build up by increasing concentration of cisplatin. And then afterwards,MTT assay, RNA interference techniques, microRNA overexpresion techniques, quantative PCR and western blotting were applied to analyze the function of CXCR4 and its downstream effectors.Result:①the expression of CXCR4 was increased in CNE2/DDP and downregulation of CXCR4 with CXCR4 siRNA was able to decrease the resistance of CNE/DDP to cisplatin; ②the expression of let-7a was decrease in CNE2/DDP, while the expression of bcl-2 was increased. Upregulation of let-7a via transfection of let-7a mimics could downregulate the expression of bcl-2 and damage the resistance of CNE2/DDP to cisplation;③downregulation of CXCR4 through CXCR4 siRNA transfection was capable of improving the expression of let-7a. Conclusion:We were the first to found that CXCR4 was related to chemoresistance of CNE2/DPP to cisplatin. Meanwhile, we confirmed that CXCR4 affected the expression of bcl-2 through regulating the expression of let-7a to modulate the chemoresistance of CNE2/DPP to cisplatin.

Association between dietary intake of folate and MTHFR and MTR genotype with risk of breast cancer.

We investigated the association between dietary intake of folate, vitamin B6, and the 5,10-methylenetetrahydrofolate reductase (MTHFR) genotype with breast cancer. A matched case-control study was conducted, and 413 patients with newly diagnosed and histologically confirmed breast cancer and 436 controls were recruited. Folate intake, vitamin B6, and vitamin B12 levels were calculated, and the MTHFR C677T and A1298C and MTR A2756G polymorphisms were analyzed by polymerase chain reaction-restriction fragment length polymorphism. Breast cancer cases were generally older, older at first live birth, and younger at menarche, had a higher body mass index, were smokers, had higher energy intake, and more first-degree relatives with breast cancer as well as more live births compared to controls. With respect to energy intake, we found that higher energy intake were more likely to increase the risk of breast cancer. The MTHFR 667TT genotype was associated with a moderately increased risk of breast cancer when compared with the CC genotype, and a significant odds ratio (OR; 95% confidence interval, CI) was found (OR = 1.70, 95%CI = 1.06-2.73). Individuals carrying T allele were associated with higher risk of breast cancer when compared with C allele (OR = 1.34, 95%CI = 1.06-1.70). We did not find a significant effect of the MTHFR A1298C and MTR A2756G on the risk of breast cancer. We did not find any association between folate intake and MTHFR C677T polymorphisms. In conclusion, we found that the MTHFR C667T polymorphism is associated with the risk of breast cancer, indicating that this genotype plays a role in breast cancer development.

Establishment of a simple living donor liver transplantation dog model using a two-step nonvenous bypass hepatectomy.

AIM: Dogs are an ideal model for studying living donor liver transplantation (LDLT). However, due to their poor tolerance to congestion and acidosis during portal vein blockage, current LDLT dog models with long operation times have high mortality. To address the issue, we designed a novel simplified operation with two-step nonvenous bypass (NVB) hepatectomy. METHODS: Eighty dogs were evenly randomized to the living liver donor (LLD) or the recipient (LLR) groups. A standard lobectomy of I, II, and III lobes was performed in the LLD group. In the LLR group, first only I, II, and III lobes were resected using NVB; the residual lobes were resected off just after donor lobes were implanted. RESULTS: For the LLD group, the operation time was 172.67 ± 20.98 minutes, amount of blood loss was 71.39 ± 13.59 mL, and 2-week survival rate was 85.00%. For the LLR group, the operation time was 251.61 ± 22.87 minutes, amount of blood loss was 220.00 ± 96.40 mL, amount of blood transfusion was 163.89 ± 44.74 mL, and 48-hour survival rate was 77.14%. In the LLR group, the mean arterial and central venous pressures decreased after organ implantion, but gradually recovered to normal levels after surgery. The liver function biochemical parameters recovered to preoperational levels after 14 days in the LLD group; in the LLR group, they gradually increased during 48 hours after operation. CONCLUSION: The present method with two-step NVB hepatectomy can be used efficiently and safely for establishing LDLT dog model.

Long-term efficacy of stereotactic bilateral anterior cingulotomy and bilateral anterior capsulotomy as a treatment for refractory obsessive-compulsive disorder.

OBJECTIVE: To investigate the long-term efficacy and adverse effects of stereotactic bilateral anterior cingulotomy (BACI) and bilateral anterior capsulotomy (BACA) as a treatment for refractory obsessive-compulsive disorder (OCD) patients. METHODS: Seven patients suffering from refractory OCD underwent stereotactic surgery and were followed for 12 months. The Yale-Brown Obsessive Compulsive Scale (Y-BOCS) was used to assess the efficacy. The test was taken before and 6 and 12 months after surgery. RESULTS: The mean Y-BOCS scores decreased significantly from 32.9 ± 4.7 at baseline to 20.6 ± 5.3 after 12 months. Five out of the 7 patients showed a decrease of more than 35%. During the 12-month follow-up, the effective rate had increased from 28.6 to 71.4%. There were no significant adverse effects observed after surgery. CONCLUSIONS: The BACI and BACA were effective for the treatment of refractory OCD, and no significant adverse effects on long-term follow-up were found.

Sleeping Beauty transposon system is a reliable gene delivery tool for hereditary tyrosinaemia type 1 disease gene therapy: size of the foreign gene decides the timing of stable integration into the host chromosomes.

OBJECTIVE: This study investigated compensation for loss of the fumaryl-acetoacetate hydrolase gene (Fah) by gene therapy using the Sleeping Beauty transposon system (SBTS), in a hereditary tyrosinaemia type 1 (HT-1) mouse model (Fah-/-). METHODS: Twenty Fah-/- study mice, five wild-type positive controls and five Fah-/- negative controls were included. All Fah-/- mice received 2-(2-nitro-4-trifluoro-methylbenzoyl)-1,3-cyclo hexaedione (NTBC). Fah-/- study mice were randomly injected with one of two SBTS constructs: Fah-SBTS (containing mouse Fah gene), or forkhead box M1b (FOXM1B)-Fah-SBTS (containing mouse Fah and human FOXM1B genes). Firefly luciferase-SBTS was injected as a trace marker. NTBC treatment stopped after construct injection; Fah-/- negative controls were kept healthy with continued NTBC. Mice were weighed daily; the luciferase signal was monitored by in vivo bioluminescence, and Fah and FOXM1B gene expression were evaluated. RESULTS: The Fah gene integrated into the mouse chromosomes within 1 week of Fah-SBTS injection (mice survived without NTBC thereafter) and within 1 month of FOXM1B-Fah-SBTS injection (mice lost weight dramatically and needed additional NTBC). CONCLUSION: The shorter Fah gene had an advantage over the longer FOXM1B-Fah gene for stable integration into the host mouse chromosomes.

Fabp3 inhibits proliferation and promotes apoptosis of embryonic myocardial cells.

Fatty acid binding protein 3 (FABP3) is a member of a family of binding proteins. The protein is mainly expressed in cardiac and skeletal muscle cells, and it has been linked to fatty acid metabolism, trafficking, and signaling. Using suppression subtractive hybridization, we previously found that FABP3 is highly regulated in ventricular septal defect (VSD) patients and may play a significant role in the development of human VSD. We therefore aimed to identify the biological characteristics of the FABP3 gene in embryonic myocardial cells. On the basis of RT-PCR and western blotting analyses, we demonstrated that the expression levels of FABP3 mRNA and protein were up-regulated initially and then gradually decreased with P19 cell differentiation. MTT assays and cell cycle analysis showed that FABP3 inhibits P19 cell proliferation, and data from annexin V-FITC assays revealed that FABP3 can promote apoptosis of P19 cells. Further data from quantitative real-time RT-PCR revealed lower expression levels of cardiac muscle-specific molecular markers (cTnT, alpha-MHC, GATA4, and MEF2c) in FABP3-overexpressing cell lines than in the control cells during differentiation. Our results demonstrate that FABP3 may be involved in the differentiation of cardiac myocytes.

Cloning, characterization and identification of Rcet1-v1 and Rcet1-v2, two novel splice variants of mouse Rcet1 related to Cres subgroup of family 2 cystatins.

Cystatins are physiological cysteine proteinase inhibitors. We used digital differential display (DDD) to clone two novel splice variants Rcet1-v1 and Rcet1-v2 which were isolated from adult mouse testis cDNA library. Sequence analysis revealed that Rcet1-v1 and Rcet1-v2 cDNAs are 454 and 610 bp in length, respectively, and each has four exons, but the lengths of their second and third exons are different, with the results that these cDNAs encoded two different putative proteins. The deduced proteins were 88 amino acid residues (RCET1-v1) and 140 residues (RCET1-v2) in length and have one potential signal peptide and one cystatin domain, respectively, but lack part critical consensus sites important for cysteine protease inhibition. These characteristics are seen in CRES subgroup, which related to the family 2 cystatins and primarily expressed in reproductive tract. RT-PCR analysis showed that Rcet1-v1 and Rcet1-v2 were specifically expressed in adult mouse testis, epididymis and cerebrum, but higher in testis than in epididymis and cerebrum. RT-PCR analysis also showed that Rcet1-v1 and Rcet1-v2 were specifically expressed in adult mouse pituitary and spermatogonium, but not expressed in spermatozoa. Results of in situ hybridization showed that Rcet1 gene expressed abundantly in mouse spermatogonium, spermatocytes and round spermatids; did not expressed in spermatozoa. At mouse testis different development stages, Rcet1-v1 and Rcet1-v2 were expressed very low from postnatal 1 day to postnatal 3 weeks; after postnatal 4 weeks, expressed steadily increased from postnatal 4 to 7 weeks, highest in postnatal 7 to 8 weeks, then keeping on the expressing level of postnatal 6 weeks in postnatal 13-57 weeks. All these indicated that Rcet1-v1 and Rcet1-v2 primarily expressed in mouse male reproductive tract and may play important roles in mouse spermatocytes and round spermatid development. Rcet1-v1 and Rcet1-v2 may be new members of Cres subgroup of the family 2 cystatins.