Mutations in the TSC2 gene: analysis of the complete coding sequence using the protein truncation test (PTT)

Mutations in the TSC2 gene on chromosome 16p13.3 are responsible for approximately 50% of familial tuberous sclerosis (TSC). The gene has 41 small exons spanning 45 kb of genomic DNA and encoding a 5.5 kb mRNA. Large germline deletions of TSC2 occur in <5% of cases, and a number of small intragenic mutations have been described. We analysed mRNA from 18 unrelated cases of TSC for TSC2 mutations using the protein truncation test (PTT). Three cases were predicted to be TSC2 mutations on the basis of linkage analysis or because a hamartoma from the patient showed loss of heterozygosity for 16p13.3 markers. Three overlapping PCR products, covering the complete coding sequence of mRNA, were generated from lymphoblastoid cell lines, translated into 35S-methionine labelled protein, and analysed by SDS-PAGE. PCR products showing PTT shifts were directly sequenced, and mutations confirmed by restriction enzyme digestion where possible. Six PTT shifts were identified. Five of these were caused by mutations predicted to produce a truncated protein: (i) a sporadic case showed a 32 bp deletion in exon 11, and a mutant mRNA without exon 11 was produced; the normal exon 10 was also spliced out; (ii) a sporadic case had a 1 bp deletion in exon 12 (1634delT); (iii) a TSC2-linked mother and daughter pair had a G-->T transversion in exon 23 (G2715T) introducing a cryptic splice site causing a 29 bp truncation of mRNA from exon 23; (iv) a sporadic case showed a 2 bp deletion in exon 36; (v) a sporadic case showed a 1 bp insertion disrupting the donor splice site of exon 37 (5007+2insA), resulting in the use of an upstream exonic cryptic splice site to cause a 29 bp truncation of mRNA from exon 37. In one case, the PTT shift was explained by in-frame splicing out of exon 10, in the presence of a normal exon 10 genomic sequence. Alternative splicing of exon 10 of the TSC2 gene may be a normal variant. Three 3rd base substitution polymorphisms were also detected during direct sequencing of PCR products. Confirmed mutations were identified in 28% of the families studied and on the assumption that half of the sporadic cases should have TSC2 mutations, a crude estimate of the detection rate would be 60%. This compares favourably with other screening methods used for TSC2, notably SSCP, and since PTT involves much less work it may be the method of choice.      

Expression of superoxide dismutase and xanthine oxidase in myometrium, fetal membranes and placenta during normal human pregnancy and parturition

Superoxide, an agent which attenuates the half-life of nitric oxide, is metabolized and synthesized by superoxide dismutase (SOD) and xanthine oxidase, respectively. Over the last few years much work has focused on the role of nitric oxide in human parturition. The aim of this study was to determine whether the onset of human parturition is associated with a change in the expression of copper/zinc superoxide dismutase (Cu/Zn SOD), manganese superoxide dismutase (Mn SOD) or xanthine oxidase within the uterus. Samples of myometrium, placenta, decidua and fetal membranes were obtained from women before and after the onset of labour at term. Immunocytochemistry was used to localize Cu/Zn SOD, Mn SOD and xanthine oxidase and measure SOD enzyme activity. Cu/Zn and Mn SOD-like immunoreactivity was detected in syncytiotrophoblast cells, villous stromal cells and endothelial cells of blood vessels in the placenta. In the myometrium Cu/Zn and Mn SOD were localized to myocytes and endothelial cells and to some vascular smooth muscle cells. In the fetal membranes we observed staining for Cu/Zn SOD and Mn SOD in the amnion, chorion, extravillous trophoblast and decidua. There was no difference in SOD enzyme activity or staining intensity for SOD between different cell types before and during labour. Xanthine oxidase immunoreactivity was identified in each of the tissues examined and again there was no difference in immunostaining in tissues obtained from women delivered before or after the onset of labour. These results show that the pregnant uterus is capable of both synthesizing and degrading superoxide and suggest that superoxide dismutase and xanthine oxidase may play a role in the maintenance of uterine quiescence during pregnancy, but not in the initiation of parturition.      

Optimization of a method for deactivation of platelet-activating factor:acetylhydrolase in serum for use in in-vitro fertilization culture media

Embryos produced by in-vitro fertilization (IVF) may produce less platelet-activating factor (PAF) than is optimal for development. It was previously shown that supplementation of culture media with PAF results in a significant increase in pregnancy rate. Human embryos are often cultured in media supplemented with serum containing the enzyme PAF:acetylhydrolase (PAF:AH; EC 3.1.1.47), which hydrolyses PAF to its inactive form, lyso-PAF. Thus, effective supplementation of media with PAF requires inactivation of this enzyme. In this study we examine the efficacy of the methods of PAF:AH deactivation used for PAF supplementation of IVF culture medium. When the effectiveness of a commonly used acid treatment protocol (pH 3.0 at room temperature for 5 min) was examined, it was found that it was not completely effective for the majority of sera. When synthetic PAF was added to 18 serum samples which had been acid treated, five had 90-100% of the original PAF remaining after 24 h (showing that the acid treatment was effective), eight had from 10-90% of the original PAF remaining after 24 h, and five samples had 0-10%. The extent to which PAF:AH was susceptible to deactivation was not associated with the activity in the serum prior to treatment, the serum oestradiol concentration, or the cause of infertility. The period of acidification and the incubation temperature were assessed to develop a new acid-treatment protocol (20 min acid treatment at 37 degrees C) which was able to deactivate PAF:AH effectively in all sera (53/53) examined. A trial was performed to assess the effect of acid treatment of serum for 5 min at room temperature compared with the new protocol (20 min at 37 degrees C) on IVF outcome, following PAF supplementation of IVF culture medium. Oocyte recovery, fertilization and embryo development rates were equivalent for both groups and approximately equal numbers of embryos were transferred or cryopreserved. Pregnancy rates were not significantly different (14.6 versus 20.0%) for the two treatments, with a trend towards a higher pregnancy rate with the new acid- treatment protocol. The results show that this new procedure for acid treatment of serum in combination with PAF supplementation does not have detrimental effects on embryos and their pregnancy outcome and is therefore suitable for use in IVF.      

Hydrosalpinges adversely affect markers of endometrial receptivity

While in-vitro fertilization (IVF) was initially developed in women with tubal factor infertility, recent clinical studies have suggested that the presence of hydrosalpinges lowers implantation and pregnancy rates. We postulated that these hydrosalpinges cause impaired endometrial receptivity. A total of 103 women with hydrosalpinges were prospectively evaluated, and compared with 55 infertile and 44 fertile controls. All women had endometrial biopsies during the window of implantation, analysed by conventional histological criteria, and also stained for three integrin markers of endometrial receptivity (alpha1beta1, alpha4beta1 and alpha vbeta3). Women with hydrosalpinges (cases) expressed significantly less of the alpha vbeta3 integrin compared with controls. There was no difference in expression of alpha1beta1 or alpha4beta1 among groups. A significantly greater number of cases had out of phase histology and missing alpha vbeta3 (type I defects) and absent integrin expression despite normal histological maturation (type II) defects, compared with controls. Of 20 women with impaired endometrial receptivity who were also biopsied after hydrosalpinx surgery, 70% demonstrated increased alpha vbeta3 expression. Seventy-seven percent of type I and 57% of type II defects were corrected postoperatively. Using markers of endometrial receptivity, this study demonstrates that inflammatory hydrosalpinges have an adverse effect on endometrial receptivity, which in some cases may be overcome by surgical treatment of the hydrosalpinx.      

大鼠视皮质胼胝体轴突的生后发育和形态──生物素结合的葡聚糖胺顺行示踪法研究

采用生物素结合的葡聚糖胺顺行示踪法研究了大鼠视皮质主要胼胝体投射区即１７／１８ａ交界区胼胝体轴突的生后发育和形态。在生后５天时，此交界区胼胝体轴突从白质向灰质Ⅰ层垂直生长，在灰质内仅有极少量的侧支抽芽。至生后１３天时，皮质Ⅰ层最先出现致密的由胼胝体轴突终支组成的终末丛。到生后１７天时，类似的终末丛也见于皮质Ⅱ／Ⅲ，Ⅴ和Ⅵ层，这种分布型式与成年大鼠者相似。以上结果表明，绝大部分胼胝体轴突首先生长到达Ⅰ层并先在Ⅰ层发出终支，然后再在其它皮质层发出侧支及终支，因而提示皮质Ⅰ层在胼胝体联系的生后发育中可能发挥重要作用。     

Positional cloning of the gene for X-linked retinitis pigmentosa 3: homology with the guanine-nucleotide-exchange factor RCC1

The gene for retinitis pigmentosa 3 (RP3), the most frequent form of X- linked RP (XLRP), has been mapped previously to a chromosome interval of less than 1000 kbp between the DXS1110 marker and the OTC locus at Xp21.1-p11.4. Employing a novel technique, YAC Representation Hybridization (YRH)', we have recently identified a small XLRP associated microdeletion in this interval, as well as several putative exons including the 3' end of a gene that was truncated by the deletion. cDNA library screening and sequencing of a cosmid centromeric to the deletion has now enabled us to identify numerous additional exons and to detect several point mutations in patients with XLRP. The predicted gene product shows homology to RCC1, the guanine-nucleotide- exchange factor (GEF) of the Ras-like GTPase Ran. Our findings suggest that we have cloned the long-sought RP3 gene, and that it may encode the GEF of a retina-specific GTP-binding protein.      

Comparison of Antibody Response Elicited by ChAdOx1 and BNT162b2 COVID-19 Vaccine

BackgroundChAdOx1 and BNT162b2 vaccines are currently commonly used against coronavirus disease 2019 worldwide. Our study was designed to determine the serostatus and relative levels of anti-S and neutralizing antibodies in patients who were administered either ChAdOx1 or BNT162b2 vaccine. In addition, we investigated whether the antibody response to each vaccine differed according to sex and age.MethodsHealthcare workers (HCWs) at a general hospital who were vaccinated with two doses of either ChAdOx1 or BNT162b2 were invited to participate in this prospective cohort study. Blood samples of HCWs vaccinated with both ChAdOx1 doses over a period of 12 weeks were collected at weeks 4 and 8 post first vaccination and 2 weeks post second vaccination. Blood samples of HCWs vaccinated with BNT162b2 were collected in the third week after the first dose, and the second dose was then administered on the same day; two weeks post second dose (5 weeks after the first dose), blood samples were collected to assess the antibody response. The titers of anti-S antibodies against the severe acute respiratory syndrome coronavirus 2 spike (S) protein receptor-binding domain and the neutralizing antibodies in the collected blood were evaluated.ResultsOf the 309 HCWs enrolled in the study, 205 received ChAdOx1 and 104 received BNT162b2. Blood samples from participants receiving either the ChAdOx1 or BNT162b2 vaccine exhibited substantial anti-S and neutralizing antibody seropositivity subsequent to the second dose. All participants (100%) from both vaccine groups were seropositive for anti-S antibody, while 98% (201/205) of ChAdOx1-vaccinated individuals and 100% (104/104) of BNT162b2-vaccinated individuals were seropositive for neutralizing antibodies. The median levels of anti-S and neutralizing antibodies were significantly higher in the BNT162b2-vaccinated group than the ChAdOx1-vaccinated group; in particular, anti-S antibody titers of 1,020 (interquartile range, 571.0–1,631.0) U/mL vs. 2,360 (1,243–2,500) U/mL, P < 0.05, were recorded for the ChAdOx1 and BNT162b2 groups, respectively, and neutralizing antibody titers of 85.0 (65.9–92.1%) vs. 95.8 (94.4–96.6%), P < 0.05, were recorded for the ChAdOx1 and BNT162b2 groups, respectively. In the ChAdOx1 vaccine group, the neutralizing antibody level was significantly higher in women than in men (85.7 [70.3–92.5%] vs. 77.7 [59.2–91.0%], P < 0.05); however, the neutralizing antibody titer in the BNT162b2 vaccine group did not vary between the two sexes (95.9 [95.2–96.6%] vs. 95.2 [93.5–96.3%], P = 0.200). Analysis of the correlation of antibody profiles with age revealed that the levels of anti-S antibodies and signal inhibition rate (SIR) of neutralizing antibodies decreased significantly with age.ConclusionBoth the ChAdOx1- and BNT162b2-vaccinated groups showed high seropositivity for anti-S and neutralizing antibodies. The SIR of neutralizing antibodies in the ChAdOx1 vaccine group was higher in women than in men. Enhanced antibody responses were observed in participants vaccinated with BNT162b2 compared to those vaccinated with the ChAdOx1 vaccine.     

Short-Ti inversion-recovery pulse sequence: analysis and initial experience in cancer imaging

Inversion recovery (IR), commonly considered a pulse sequence capable of producing T1-weighted images with excellent display of normal anatomy, is versatile: The null point and peak time provide a useful, succinct summary of the properties of IR and its capacity for producing both T1- and T2-weighted images. Shortening of the inversion time (TI) and creation of a short-TI inversion-recovery (STIR) pulse sequence increases sensitivity to malignancy and other abnormalities by making the effects of prolonged T1 and T2 on signal intensity additive and by nulling the signal from fat. The authors examined over 300 patients with various malignancies and compared STIR images with T1- and T2-weighted images obtained at 0.5 T. In 43 cases, signal-difference-to-noise ratios (SD/Ns) were calculated between tumor, fat, and muscle. In general, STIR images demonstrated tumor as a conspicuously high-intensity area in a background of muted, discernible anatomic detail. The good contrast achieved with STIR sequences between tumor and fat (SD/N = 18.1) and tumor and muscle (SD/N = 12.9) consolidated into a single image the information contained separately on T1- and T2-weighted images, which facilitates efficient detection and localization of malignancy.