Rapid combined genotyping of factor V, prothrombin and methylenetetrahydrofolate reductase single nucleotide polymorphisms using minor groove binding DNA oligonucleotides (MGB probes) and real-time polymerase chain reaction.

Risk factors for cardiovascular diseases and venous thromboembolism involve both acquired and hereditary conditions. Among the latter, mutations in genes coding for coagulation factors (factor V Leiden [Arg506Gly], G20210A in the 3'-untranslated region of factor II ) and variant C677T of the methylenetetrahydrofolate reductase (MTHFR ) are often involved and co-inherited. These three factors were genotyped simultaneously in the same 96-well plate, using a real-time polymerase chain reaction (PCR) Taqman assay and minor groove binding DNA oligonucleotides (MGB probes). While primers and MGB probes matched their corresponding single nucleotide polymorphism (SNP), the real-time MGB program was identical for each target gene. Homozygous wild-type (WT; -/-), heterozygous (+/-) or homozygous (+/+) variants (n = 362) were selected for factor V (n = 115, with -/-, 40; +/-, 40; +/+, 35), factor II (n = 122, with -/-, 60; +/-, 60; +/+, 2), and MTHFR (n = 120, with -/-, 40; +/-, 40; +/+, 40), according to the results of conventional PCR-restriction fragment length polymorphism (PCR-RFLP), but the allelic discrimination was performed blind. Results of the real-time MGB and PCR-RFLP assays were identical. This new assay was easy and fast with high throughput, without risk of molecular carryover, and cost-effective for laboratories utilizing the Taqman or related fluorescence reading methods. These advantages make it particularly suitable for large-scale combined genotyping of several polymorphisms in the routine setting.     

Radiographic assessment of instability of the knee due to rupture of the anterior cruciate ligament. A quadriceps-contraction technique

We compared the results of a radiographic technique for the measurement of instability of the knee with those obtained with a KT-1000 arthrometer. The study was conducted on both knees of sixty patients who had a ruptured anterior-cruciate ligament in one knee, as well as in ten control subjects. The radiographic technique included the examination of a true lateral radiograph, made while the knee was in full extension and the quadriceps was maximally contracted, with a 66.7-newton downward force produced by a 6.8-kilogram weight suspended from the ankle. As demonstrated by both techniques, the maximum difference between the displacements of the right and left knees in the control subjects was 2.5 millimeters and the mean difference between the displacements in the two knees in the patients was 7.5 millimeters. In fourteen of the sixty knees in which the ligament was ruptured, the injury was acute. The forward translation of the medial side in these fourteen knees was compared with that in the forty-six knees in which the injury was chronic. The mean difference in the displacement of the medial side in the right and left knees was 3.5 millimeters in the fourteen patients who had an acute injury and 5.0 millimeters in the forty-six patients who had a chronic injury. Thirteen of the sixty patients had disruption of the posteromedial corner of the injured knee, and the translation of the medial side in these knees was significantly increased compared with that in the intact knees of the same patients.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of ami-25 on liver vessels and tumors on T1-weighted turbo-field-echo images: Implications for tumor characterization

This study was devoted to tumor differentiation in liver MR T1-weighted imaging with superparamagnetic iron oxide (SPIO). Twenty-one patients with 40 liver lesions were studied at 1.5 T. Before and at least 45 minutes after SPIO administration, turbo-field-echo (TFE) T1-weighted, TFE T1 × T2*-weighted (MXT), and fat-suppressed turbo-spin-echo T2-weighted images were acquired. A quantitative analysis was performed blindly. On TFE T1-weighted images, the signal enhancement was ?33% ± 12 for the liver, ?24% ± 2 for adenomas and focal nodular hyperplasia, +60% ± 33 for the hemangiomas; metastases and cyst enhancement were not significant. After SPIO on TFE T1-weighted images, the hemangioma-to-liver signal ratio (149% ± 18) was definitely higher than the mean metastasis-to-liver signal ratio (90% ± 16). This T1-related differentiation ability lacked dramatically on TFE MXT images and, in one case, was reduced on post-SPIO TFE T1-weighted images by a long imaging delay after SPIO administration (2 hours).     

Endothelin-1 in children with chronic renal failure

Endothelin-1 (ET-1) was meansured after extraction from plasma of normal adults (5.9±1.9 pg/ml,n=22), normal children (7.1±1.86 pg/ml,n=29), nonhaemodialysed children with chronic renal failure (CRF) (11.1±1.8 pg/ml),n=10), renal graft recipients (9.5±3.4 pg/ml,n=37), haemodialysed children 24 h after a haemodialysis session (20.02±10.9 pg/ml,n=26) and haemodialysed children before and after a haemodialysis session (15.31±10.6 and 13.8±8.5 respectively,n=14). A sensitive and specific radioimmunoassay was used. ET-1 was significantly higher in non-haemodialysed CRF children and in renal graft recipients than in normal children (P<0.001 andP<0.01, respectively) and significantly higher in haemodialysed children when compared with normal children, non-haemodialysed CRF children and renal graft recipients (P<0.001). ET-1 concentrations were similar in normal children and normal adults. ET-1 was inversely correlated with glomerular filtration rate in non-haemodialysed CRF children (r=–0.39,P<0.01) and positively correlated with extracellular volume in haemodialysed children (r=0.435,P<0.03). After haemodialysis, ET-1 increased in 6 and decreased in 8 of the 14 children studied before and after a haemodialysis session.     

Human ovarian granulosa cells and follicular fluid indices: the relationship to oocyte maturity and fertilization in vitro

The study investigates the correlation between oocyte maturity and fertilization and a variety of hormonal parameters in follicular fluid and ovarian granulosa cells. A methodology for purification of granulosa cells from contaminating blood cells is also established. A total of 63 follicular aspirates were collected at oocyte retrieval from 30 women superovulated using the long luteinizing hormone- releasing hormone (LHRH analogue)/human menopausal gonadotrophin regimen. Oestradiol, progesterone, testosterone and human chorionic gonadotrophin (HCG) were quantified in follicular fluid and granulosa cells were immunostained for human chorionic gonadotrophin. Immunopurification of granulosa cells from contaminating blood cells was performed. HCG in follicular fluid was significantly high in follicles yielding immature (grade 3) oocytes (P=0.002); there was no correlation with fertilization. Aspirates from follicles containing mature (grade 1) oocytes and oocytes that subsequently fertilized had significantly more granulosa cells immunobound to HCG (P < 0.001, P=0.02). Moreover, the immunomagnetic purification technique provided >98% pure population of granulosa cells. The data demonstrate that HCG in follicular fluid and on granulosa cells may help to predict oocyte maturity and fertilization. Furthermore, immunomagnetic beads provide a reliable procedure for the purification of ovarian granulosa cells.      

Structure and function in urinary bladder of foetal sheep

1. The structure and function of the epithelial lining of the urinary bladder of sheep foetuses was investigated by electron microscopic studies made in conjunction with a series of experiments in which the permeability of the bladder to sodium and water was measured in vitro. Measurements were made at gestational ages ranging from 50 to 141 days (term = 147 days) Osmolarity and electrolyte concentrations of urine found in the foetal bladder were also measured.2. The development of tight junctions between the bladder epithelial cells was investigated by incubating the tissue with solutions containing 1 mM-LaCl(3) on the mucosal surface. No penetration of the junctions by lanthanum was observed in foetuses of 90 days or older. In younger bladders, the epithelial layer was stripped by treatment with lanthanum, but tight junctions appeared to be fully developed in early bladders incubated without lanthanum.3. The surface structure of the luminal (mucosal) plasmalemma was fully developed at 50 days.4. Unidirectional fluxes of labelled sodium and water were measured with identical solutions bathing the two surfaces of the bladder wall. No net water movement occurred; the mean ratio of efflux to influx in nine bladders was 1.002 +/- 0.039 (S.E. of mean). Under these conditions, the flux ratio for sodium was 1.735 +/- 0.143 (S.E. of mean) in twelve bladders.5. Antidiuretic hormone (ADH) had no effect on net water movement but reduced the net efflux of sodium so that the flux ratio became 1.285 +/- 0.255 (S.E. of mean) n = 8. ADH also had a striking effect on the structure of the epithelium, causing marked swelling of the intercellular spaces. The tight junctions remained an effective barrier to lanthanum penetration under these conditions; lanthanum was not observed in the enlarged spaces.