RHD mRNA替代剪接区域研究

目的2007年国内报道一例弱D型个体存在第4—9外显子选择性剪接的转录子,我们探讨正常Rh（D）阳性个体的RHD基因mRNA的选择性剪接区域。方法随机选择3名Rh（D）阳性个体,从新鲜全血中提取总RNA,通过特异性引物,采用“一步法”逆转录-PCR（1iT—PCR）,扩增RHDmRNA第1～7外显子区域,以及第6-10外显子区域,然后cDNA琼脂糖凝胶电泳和成像分析。结果未发现第1～7外显子区域存在mRNA的选择性剪接条带,仅存在由特异性引物所扩增的第1—7外显子全长的序列条带;而第6～10外显子区域观察到5种替代剪切条带,序列分析显示分别为无缺失片段,以及完整缺失第7、第9、第7和9、第7—9外显子5种RHD转录子。结论正常Rh（D）抗原阳性个体的RHD基因mRNA的选择性剪接仅存在于第7～9外显子区域。     

人RHD基因的克隆和鉴定

目的克隆人RHD基因,并对其进行鉴定。方法以RhD阳性志愿者骨髓为材料,用TRIzol试剂提取总RNA;设计、合成人RHD基因扩增引物,RT-PCR方法扩增RHD基因片段;T／A克隆后将其亚克隆人pET28a（＋）载体中,经酶切、PCR和测序对重组质粒进行鉴定。结果骨髓总RNA被成功提取;RT-PCR成功扩增出RHD基因片段,其大小与预期约509bp基本一致;T／A克隆后再将其亚克隆,通过酶切和PCR证明RHD基因成功亚克隆入pET28a（＋）载体中;基因测序结果比对显示,与已公布的RHD基因（GenBank登录号为NM016124）序列基本一致,同源性为98％。结论成功克隆了RHD基因,这将为进一步研究奠定基础。     

Is fetal testis in danger?

Estrogens are classically known to play a major role in female reproduction, but there is now compelling evidence that they may also be involved in the regulation of male reproductive function. In humans, a decrease in sperm count and an increase in the incidences of testicular cancer, cryptorchidism and hypospadia have been observed in many countries over the last 50 years. Male reproductive alterations were also observed in wildlife. Such male reproductive disorders have been attributed to the increase in concentration of xenobiotics, and of xenoestrogens in particular, in the environment and in food. Epidemiological, clinical and experimental studies have suggested that excessive exposure to estrogens during fetal/neonatal life can lead to reproductive disorders in adulthood. Using an in vitro model, we showed that estrogens directly affected the development of the fetal testis. Lastly, we clearly demonstrated that the fetal and neonatal testis is very sensitive to estrogens since the invalidation of estrogen receptor alpha leads to an increase of steroidogenesis and the invalidation of estrogen receptor beta enhances the development of the germ cell lineage in the male.     

鉴定9个新的RHD基因mRNA可变剪接体

为了研究各种RHD基因mRNA可变剪接体的基因结构, 应用逆转录聚合酶链反应(RT-PCR)检测正常人脐血样本RHD mRNA, 对RHD cDNA进行TA克隆和序列分析, 对各可变剪接体的剪接位点进行DNA序列分析, 并将RHD mRNA进行表达序列标签(ESTs)分析。结果在28个阳性克隆中, 除全长RHD cDNA外, 共检测到12种(包括9种新的)RHD可变剪接体, 发现外显子遗漏、5′和3′剪接位点变异3种剪接形式, 涉及外显子2~9, 其中6种新的剪接体同时存在RHD和RHCE基因同源杂交现象。ESTs分析还检索到内含子保留形式的剪接体。研究表明, RHD基因mRNA存在复杂的可变剪接机制, 除已报道的剪接体外, 检测到9种新的RHD可变剪接体, 并发现了可变剪接和同源杂交并存现象。     

Does fetal sex affect pregnancy outcome?

Background: In maternal fetal medicine, gender differences in outcome are often observed.Objective: This article reviews the fetal sex-dependent differences found in many aspects of pregnancy, from conception through birth.Methods: The MEDLINE, EMBASE, and Current Contents databases were searched, for the years 1985 to 2006, using the following Medical Subject Headings and text words: fetal gender, finale, female, sex ratio at birth, pregnancy outcome, preterm birth, and stillbirth. The search was not limited by language. In addition, the bibliographies of known relevant articles were examined to capture any reports not already identified in the electronic search. All reports that provided information on gender differences in pregnancy outcome were included for review.Results: An extremely high male-to-female ratio was found in fetuses born after very short-duration pregnancy; this level declined around the 20th week and stabilized at term. In the absence of manipulation, both the sex ratio at birth and the population sex ratio have been found to remain consistent. A higher incidence of preterm birth and premature preterm rupture of membranes has been observed in different populations among mothers of male newborns compared with mothers of females. It has been speculated that this higher incidence may be linked to the relatively greater weight at lower gestational age of male newborns versus females. Women carrying male fetuses had higher rates of gestational diabetes mellitus, fetal macrosomia, failure to progress during the first and second stages of labor, cord prolapse, nuchal cord, and true umbilical cord knots. Cesarean sections were also more frequently found among male neonates compared with females.Conclusions: Male sex is an independent risk factor for adverse pregnancy outcome. Evidence suggests that females have an advantage over males, with a better outcome in the perinatal period, particularly after preterm birth.Key words: fetal gender, male, female, sex ratio of birth, perinatal outcome.     

Dendritic cells: key to fetal tolerance?

Pregnancy is a unique event in which a fetus, despite being genetically and immunologically different from the mother (a hemi-allograft), develops in the uterus. Successful pregnancy implies avoidance of rejection by the maternal immune system. Fetal and maternal immune cells come into direct contact at the decidua, which is a highly specialized mucous membrane that plays a key role in fetal tolerance. Uterine dendritic cells (DC) within the decidua have been implicated in pregnancy maintenance. DC serve as antigen-presenting cells with the unique ability to induce primary immune responses. Just as lymphocytes comprise different subsets, DC subsets have been identified that differentially control lymphocyte function. DC may also act to induce immunologic tolerance and regulation of T cell-mediated immunity. Current understanding of DC immunobiology within the context of mammalian fetal-maternal tolerance is reviewed and discussed herein.     

Role of α-glucosidase in fetal lung maturation

The role of lysosomal enzyme acid α-glucosidase in fetal lung development was investigated with the aid of a specific inhibitor, the pseudosaccharide acarbose. The drug was added to a Waymouth culture medium of fetal rat lung expiants cultivated for 48 h from gestational stage 19.5 days, an in vitro system previously shown to allow morphological and biochemical maturation of alveolar epithelium. Glycogenolysis was reduced by 40% as compared with tissue cultivated on control medium, which means that α-glucosidase could account for as much as 40% of fetal lung glycogenolysis, the remaining 60% being presumably achieved by cytosolic phosphorylase and by a microsomal neutral α-glucosidase. By the same time, the increase of phospholipids of surfactant fraction extracted from cultivated expiants was partially inhibited: total and saturated phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol and phosphatidylinositol were about 30–40% lower than in lungs cultivated on control medium. It should be emphasized that DNA concentration and increases in non-surfactant phospholipids were unchanged by the drug. α-Glucosidase activity was evidenced in the lysosomal fraction, in the microsomal fraction and, although in lower amounts, in the surfactant fraction extracted from term fetal lung. The results suggest that lysosomal α-glucosidase plays a major role in lung maturation and could facilitate glycogenolysis for the specific use of glycogen stores in providing substrates for surfactant phospholipid biosynthesis.     

Bilateral fetal uropathy: what is the outlook?

OBJECTIVE--To assess the morbidity and mortality associated with a prenatal diagnosis of bilateral fetal uropathy. DESIGN--Retrospective study. SETTING--Departments of radiology, paediatric surgery, obstetrics, and pathology in two teaching hospitals that serve as referral centres for the Yorkshire region. PATIENTS--126 Cases of fetal uropathy were referred either prenatally or postnatally from hospitals in Yorkshire between August 1982 and December 1987. The disease was bilateral in 54 cases and unilateral in 72 cases. In 14 cases bilateral fetal uropathy was associated with coexistent disease. INTERVENTIONS--All cases were managed individually by an obstetrician after discussion with the radiologists and paediatric surgeons. Babies who survived were treated prophylactically with antibiotics after delivery and were operated on if appropriate. END POINT--Assessment of prognosis for long term renal function for each baby referred between August 1982 and December 1987: follow up ranged from six months to five years. MEASUREMENTS AND MAIN RESULTS--Of the 54 fetuses with bilateral fetal uropathy, 13 were terminated as the prenatal findings of ultrasonography were considered to be incompatible with long term survival. Ten of the liveborn babies died, five of renal or pulmonary insufficiency, or both, and five of associated congenital anomalies. Thirty one infants survived to follow up; four of these had serious coexistent disease and two had impaired renal function. Thus the overall mortality was 43% and the morbidity rate 19%. The renal anomaly was associated with other serious disease in 14 cases (26%) compared with two (3%) of the 72 cases of unilateral fetal uropathy. All but two of the 27 infants with isolated bilateral urinary tract disease had excellent prospects for survival. CONCLUSION--Although bilateral fetal uropathy is associated with a high morbidity rate and mortality, careful prenatal assessment can help to identify fetuses with a poor prognosis. The outlook for a fetus with isolated renal disease if treated promptly after delivery is excellent and compares favourably with that reported after prenatal surgical intervention.     

Transforming growth factor β3 in the fetal and neonatal rat testis: immunolocalization and effect on fetal Leydig cell function

The localization of transforming growth factor β3 (TGFβ3) in the fetal and neonatal testis (from fetal day 13.5 to postnatal day 6) was investigated by immunohistochemical staining with a specific polyclonal antibody raised against a synthetic peptide corresponding to residues 50–75 of TGFβ3. This antibody recognized 0.5 ng TGFβ3 in western blot analysis, but did not detect 25 ng TGFβ1 or TGFβ2. The immunolocalization of TGFβ3 in the fetal and neonatal testis changed throughout development. Immunostaining was present in the gonocytes by fetal day 13.5, persisted until postnatal day 3, and was heterogeneous in spermatogonia on postnatal day 6. The Sertoli cells contained no immunoreactivity at any age. The fetal-type Leydig cells were first immunostained for TGFβ3 on day 16.5 and staining became very intense from day 18.5 onward. Staining disappeared when the antibody was presaturated with the synthetic peptide, but persisted when the antibody was presaturated with a tenfold excess of the corresponding peptide from TGFβ2. Furthermore, we researched whether TGFβ3 could act as a local regulator of fetal Leydig cell function. In a dispersed fetal testicular cell system, TGFβ3 inhibited the LH-stimulated testosterone production by Leydig cells from 20.5-day-old fetuses. The inhibitory effect of TGFβ3 was equal to that observed with TGFβ1 or TGFβ2. When compared with our previous studies showing the immunolocalization of TGFβ1 and TGFβ2, the present study shows that TGFβ3 may have a specific role in the developing rat testis, but may also overlap the action of TGFβ1 and TGFβ2. Accepted: 8 June 1999     

CEUSPEC — a computerized system for fetal home telemetry

We have developed a computerized system whereby the fetal heart rate can be recorded telemetrically from patients' homes, transmitted over conventional public telephone lines, and then computer-processed in real time in the obstetric unit.     

Do male germ cells begin meiosis during fetal life?]

The existence of a preleptotene chromosome condensation and decondensation stage occuring between the last premeiotic interphase and the leptotene stage was described in numberous plants. This stage was also reported in the human fetal oocyte and in various animals (rabbit, sheep, mouse). A similar process of chromosome condensation was described in the human foetal testis, but in this latter, the decondensation phase leading to leptotene was never observed. According to this observation and to various experimental results from the literature, the preleptotene condensation stage could be related to the processes of meiotic initiation. It could represent a phase of transition between mitotic and meiotic behaviour during which the germinal cell could be sensitive to meiosis-inducing factors. It is suggested that the male germinal cell 46,XY could enter meiosis. This hypothesis is confirmed by the observation of the capacity of the XY germ cell in the mouse to become an oocyte. This capacity is normally not expressed, due to the repressive control of the adjacent Sertoli cells. Thus, stimulation of the germinal cell to enter meiosis could result from environmental factors rather than from a genetic programmation.     

The vγ chain of fetal hemoglobin of the orangutan

Previous studies suggested that orangutans have nonallelic structural genes for their γ chains, because either a threonyl or an alanyl residue may occupy position 135. Further investigation has now detected that position 75 may have either an isoleucyl or a valyl residue. From available evidence, the isoleucyl and threonyl residues are in one chain and the valyl and alanyl in the other. Orangutans appear to be homozygous for the two types of nonallelic genes.     

Does the fetal genotype affect maternal physiology during pregnancy?

Conventional wisdom states that associations between fetal growth and diseases in pregnancy, such as pregnancy-induced hypertension (PIH) and gestational diabetes (GDM), result from effects of the mother's genotype or environment acting on her physiology which subsequently affect the fetus. However, recent evidence from human mothers carrying macrosomic offspring with Beckwith Wiedemann syndrome and pregnant mice carrying p57(kip2)-null offspring suggest that variation in the fetal genome can modify maternal physiology to increase fetal nutrient delivery and optimise growth. These are some of the first documented examples of such effects, whereby the genome of one individual directly affects the physiology of another related individual from the same species. We propose that this mechanism is involved in the aetiology of PIH and GDM.     

The effect of fetal hypophysectomy and fetal death in gilts with small litters on concentrations of relaxin,progesterone and estradiol-17β

During late pregnancy concentrations of relaxin, progesterone (P) and estradiol-17β (E) in maternal plasma were measured in gilts with small litters of intact, hypophysectomized, partially hypophysectomized or dead fetuses and in gilts with litters of normal fetuses and numbers. To achieve a small litter size at parturition all but one or two fetuses were killed at surgery at Day 30 to 40 of gestation. Fetal hypophysectomy or sham procedures were attempted on Day 90 to 95. Gestation was prolonged in gilts carrying hypophysectomized or partially hypophysectomized fetuses (P<0.01). Lactation and farrowing did not occur if hypophysectomy was complete. Basal concentrations of E in plasma were lower (P<0.01), basal P appeared higher and basal relaxin was unchanged in gilts carrying hypophysectomized or dead fetuses as compared to gilts with intact fetuses. Near the end of pregnancy the concentration of E was 119.8 pg/ml in gilts with the normal number of fetuses, 32.6 pg/ml in the group with hypophysectomized fetuses, and 7.3 pg/ml in gilts with dead fetuses. The relaxin peak occurred near term in control pigs and was delayed in the groups with hypophysectomized and partially hypophysectomized fetuses. The concentration of relaxin at the peak in gilts with normal sized litters was 181.4±75.8 ng/ml as compared with 25.3±16.0 ng/ml in gilts with partially hypophysectomized fetuses and was 9.5±1.4 ng/ml in gilts with hypophysectomized fetuses and 10.6±3.3 ng/ml in gilts with one or two fetuses. In gilts with intact or partially hypophysectomized fetuses, or litters containing both types, which came into labor, the patterns of P and E were similar. In gilts with hypophysectomized fetuses, P and E at term showed little change from basal concentrations. The results confirm that the fetus influences basal concentrations of E and possibly P in late normal gestations. In addition, the presence of the fetal pituitary is associated with the peak in relaxin expected at term. These associations are likely to be related to pituitary function and/or the mass of the conceptus. Fetal hypophysectomy is clearly associated with maternal concentrations of P and E at Day 114 that are different from those in normal sows, suggesting that these two hormones may have an effect on the initiation of parturition in the pig.     

Purification of 20α-hydroxysteroid oxidoreductase from bovine fetal erythrocytes

NADPH-dependent 20α-hydroxysteroid oxidoreductase (20α-HSD; EC 1.1.1.149) from bovine fetal erythrocytes was obtained for the first time free of hemoglobin by a new 2,500-fold purification scheme. This was achieved by a sequence of calcium phosphate gel adsorption, ammonium sulfate fractionation, and affinity chromatography. The present results lead us to believe that the NADPH-dependent 3β-hydroxysteroid oxidoreductase activity, which was co-purified with 20α-activity, may originate at the active site of 20α-HSD (2).     

基于混沌游走方法的Rh血型系统中RHD基因的分析

利用基于经典HP模型的蛋白质序列混沌游走方法（chaos game representation,CGR）,给出了RHD基因的蛋白质序列CGR图,可视作蛋白质序列二级结构的一个特征图谱描述．对临床上的血型鉴别有一定的参考价值．另外．还根据由Jeffrey在1990年提出的描绘DNA序列的CGR方法,给出了RHD基因的DNA序列的CGR图．并且根据RHD基因DNA序列的CGR图算出了尺日D基因相应的马尔可夫两步转移概率矩阵,从概率矩阵表可以看出RHD基因对编码氨基酸的三联子的第3个碱基的使用偏好性．     

Cryopreserved human fetal pancreas: a source of insulin-producing tissue?

Human fetal pancreata (HFP) were obtained from dilatation and extraction aborted fetuses of 11-18 weeks' gestation. The pancreas was excised under sterile conditions and kept in culture medium at 4 degrees C, prior to stepwise digestion into 50- to 150-micron fragments. The fragmented pieces were allowed to sediment by gravity, then transferred to tissue culture for 24-48 h, and cryopreserved. The freeze-thaw protocol used stepwise equilibration with dimethyl sulfoxide, nucleation of the sample at -10 degrees C, and a slow cooling rate of 0.25 degrees C/min to -40 degrees C, followed by submersion in liquid nitrogen (-196 degrees C). Rapid thawing at 300 degrees C/min from -196 degrees C was employed. Both fresh and frozen-thawed HFP fragments appeared viable as judged by light and electron microscopy, and secreted insulin in a perifusion system upon stimulation with glucose (28 mM) and theophylline (10 mM) or glucose (2.8 mM) and theophylline (10 mM). Six patients with Type I insulin-dependent diabetes mellitus, already requiring immunosuppression for a kidney transplant, had intraportal injection of 20 cryopreserved-thawed and pooled HFP fragments. Up to the 1-year post-transplant follow-up, there has been no evidence of in vivo insulin or C-peptide production. The usefulness of cryopreserved human fetal pancreata as a source of insulin-producing tissue for diabetic patients, therefore, remains to be demonstrated.     

Glycolytic enzyme binding in fetal brain — The role of actin

The binding of aldolase, glyceraldehyde-3-phosphate dehydrogenase and pyruvate kinase in fetal calf brain homogenates and extracts has been investigated at both 0° and 37°C under high ionic strength conditions. The results demonstrate far greater enzyme binding at 37°C than at 0°C, which correlates with an increased sedimentation of cytoskeletal actin at the higher temperature. A dependence of enzyme sedimentation on the presence of polymerised actin was also demonstrated, and this indicates that cytoskeletal actin is a major adsorbent of glycolytic enzymes in this non-muscle tissue.