Rapid multiplex analysis for the factor V Leiden and prothrombin G20210A mutations associated with hereditary thrombophilia

Thromboembolic episodes are common events and affect approximately one in 1,000 persons annually. Pulmonary embolism alone accounts for 50,000 to 100,000 deaths per year in the United States with > 50% of those being elderly persons. Resistance to activated protein C is the most common inherited disorder associated with hereditary thrombophilia. A missense mutation has been identified in the gene coding for coagulation factor V (codon 506) which renders this procoagulant factor resistant to inactivation by activated protein C resulting in an increased risk for venous thrombosis. Recently, a second polymorphism was identified in the prothrombin gene (factor II) which is also associated with increased risk for venous thrombosis. Because of the high prevalence of these two mutations in the general population as well as in specific patient populations, the ability readily to detect these two mutations must be feasible. In this study, we evaluated 303 patients for the prothrombin mutatin (G20210A) which were previously tested for the factor V mutation using established polymerase chain reaction-mediated restriction fragment length polymorphism assays. In these patients, 30 (9.9%) were found to be heterozygous for the factor V Leiden mutation with no homozygous mutants identified. Twenty individuals (6.6%) were heterozygous for the prothrombin G20210A mutation, and we identified two individuals (0.66%) who were homozygous for the 20210A allele. Of the total 303 individuals screened, two were double heterozygotes for both the factor V Leiden and the prothrombin gene mutations. We also describe a multiplex polymerase chain reaction-mediated restriction fragment length polymorphism assay for detecting both mutations in a single-tube double-enzyme digestion reaction making identification of these two mutations easily achievable.     

Phase I trial of weekly scheduling and pharmacokinetics of titanocene dichloride in patients with advanced cancer

PURPOSE: To determine the maximum-tolerated dose (MTD) and the dose-limiting toxicities (DLTs) of a weekly schedule of titanocene dichloride (TD) and to define the pharmacokinetics of titanium in plasma and urine. PATIENTS AND METHODS: Twenty patients with a median age of 58 years received 83 courses of TD. TD was given as 1-hour infusion at escalating doses from 70 to 185 mg/m2/wk. Pharmacokinetic analysis was performed in eight patients for total plasma titanium (TPTi) and in three patients for ultrafiltrable titanium (UFTi). RESULTS: At the fifth dose level (185 mg/m2/wk), a variety of DLTs were seen in five patients: fatigue in three, bilirubinemia in one, and hypokalemia in two. A further six patients were treated at 140 mg/m2; only one had dose-limiting creatinine elevation and this dose was therefore defined as the MTD. No myelosuppression or alopecia were observed. One patient with adenocarcinoma of unknown primary had a minor response. Pharmacokinetic analysis showed that TPTi maximum concentration (Cmax) values were linear with dose and elimination of TPTi was triphasic with a long terminal half-life (t1/2; median, 165 hours; range, 89 to 592). Between 7% and 24.3% of the total of administered titanium was eliminated in urine over the first 24 hours. In contrast, UFTi elimination was described by a one-compartment model with a t1/2 of 0.41 hours; peak levels of UFTi were 5.2% +/- 2.5% those of TPTi. CONCLUSION: The MTD of TD given on a weekly schedule is 140 mg/m2, with cumulative, but reversible creatinine and bilirubin elevation being the DLTs.     

Targeting peptide nucleic acid-protein conjugates to structural features within duplex DNA

URF13 is the product of a mitochondrial-encoded gene (T-urf13) found only in maize plants containing the Texas male-sterile cytoplasm (cms-T), and it is thought to be responsible for both cytoplasmic male sterility and the susceptibility of cms-T maize to the fungal pathogens Bipolaris maydis race T and Phyllosticata maydis. Mitochondria isolated from cms-T maize are uniquely sensitive to pathotoxins (T-toxin) produced by these fungi and to methomyl (a commercial insecticide). URF13 acts as a receptor that specifically binds T-toxin to produce hydrophilic pores in the inner mitochondrial membrane. When expressed in Escherichia coli cells, URF13 also forms hydrophilic pores in the plasma membrane if exposed to T-toxin or methomyl. Topological studies established that URF13 contains three membrane-spanning alpha-helices, two of which are amphipathic and can contribute to pore formation. Chemical cross-linking of URF13 was used to demonstrate the existence of URF13 oligomers in cms-T mitochondria and E. coli cells. The ability of the carboxylate-specific reagent, N,N'-dicyclohexycarbodiimide, to cross-link URF13 was used in conjunction with site-directed mutagenesis to establish that the URF13 tetramer has a central core consisting of a four-alpha-helical bundle which undergoes a conformational change after interaction with T-toxin or methomyl. Overall, the experimental evidence indicates that URF13 functions as a ligand-gated, pore-forming T-toxin receptor in cms-T mitochondria.     

Singularity Analysis of Lower Mobility Parallel Manipulators Using Grassmann–Cayley Algebra

This paper introduces a methodology to analyze geometrically the singularities of manipulators, of which legs apply both actuation forces and constraint moments to their moving platform. Lower mobility parallel manipulators and parallel manipulators, of which some legs have no spherical joint, are such manipulators. The geometric conditions associated with the dependency of six PlUumlcker vectors of finite lines or lines at infinity constituting the rows of the inverse Jacobian matrix are formulated using Grassmann-Cayley algebra (GCA). Accordingly, the singularity conditions are obtained in vector form. This study is illustrated with the singularity analysis of four manipulators.     

Three-year outcome after balloon aortic valvuloplasty. Insights into prognosis of valvular aortic stenosis

BACKGROUND: To identify predictors of long-term outcome after balloon aortic valvuloplasty, we analyzed data on 674 adults (mean age, 78 +/- 9 years; 56% were women) undergoing this procedure at 24 clinical centers who had a mean initial increase in aortic valve area of 0.3 cm2. METHODS AND RESULTS: Baseline data included clinical, echocardiographic, and catheterization variables. Follow-up data included mortality, cause of death, rehospitalization, 6-month echocardiography, and functional status. Kaplan-Meier curves and log-rank tests were used to evaluate survival in subgroups. Multivariate Cox regression models were used to identify independent predictors of survival. Overall survival was 55% at 1 year, 35% at 2 years, and 23% at 3 years, with the majority of deaths (70%) classified as cardiac by an independent review committee. Rehospitalization was common (64%), although 61% of survivors at 2 years reported improved symptoms. Echocardiography at 6 months (n = 115) showed restenosis from the postprocedural valve area of 0.78 +/- 0.31 cm2 to 0.65 +/- 0.25 cm2 (P < .0001). With stepwise multivariate analysis, sequentially adding clinical, echocardiographic, and catheterization variables, the overall model identified independent predictors of survival as baseline functional status, baseline cardiac output, renal function, cachexia, female gender, left ventricular systolic function, and mitral regurgitation. Baseline and postprocedural variables were examined to identify which subgroup of patients has the best outcome after aortic valvuloplasty. A "lower-risk" subgroup (28% of the study population), defined by normal left ventricular systolic function and mild clinical functional limitation, had a 3-year survival of 36% compared with 17% in the remainder of the study group. CONCLUSIONS: Long-term survival after balloon aortic valvuloplasty is poor with 1- and 3-year survival rates of 55% and 23%, respectively. Although survivors report fewer symptoms, early restenosis and recurrent hospitalization are common.     

The effect of combined antenatal vitamin K and phenobarbital therapy on umbilical blood coagulation studies in infants less than 34 weeks' gestation

OBJECTIVE: To determine if antenatal vitamin K and phenobarbital therapy affect coagulation studies in umbilical blood at birth, and to provide 95% reference ranges for umbilical blood coagulation parameters in premature gestations. METHODS: Patients at imminent risk for spontaneous or indicated premature delivery less than 34 weeks' gestation were randomized to receive either placebo or vitamin K and phenobarbital. Prothrombin time (PT), activated partial thromboplastin time (PTT), functional coagulation factors, and decarboxylated prothrombin assays were performed on umbilical blood specimens. Decarboxylated prothrombin, also known as "protein induced by vitamin K absence-factor II" or precursor prothrombin, is a sensitive marker for vitamin K deficiency. Standardized values of PT and PTT are reported in seconds and standardized values of factor assays in percentage of normal adult functional activity (mean +/- one standard deviation). RESULTS: Newborns in the placebo and treatment groups had similar umbilical blood PT (12.6 +/- 1.2 versus 12.7 +/- 1.4 seconds), PTT (48.8 +/- 13.4 versus 49.6 +/- 13.8 seconds), and functional activity of factor II (40.3 +/- 12.5 versus 42.0 +/- 12.1%), factor VII (67.0 +/- 20.9 versus 66.8 +/- 18.9%), factor IX (27.4 +/- 12.8 versus 25.8 +/- 8.9%), and factor X (47.0 +/- 12.8 versus 49.2 +/- 11.6%). Newborns in the treatment group were about half as likely as those in the placebo group to have detectable decarboxylated prothrombin levels in umbilical blood at birth (gestational age-adjusted odds ratio 0.47, 95% confidence interval 0.22-1.01; P = .05). CONCLUSIONS: Combined maternal therapy with vitamin K and phenobarbital before premature delivery does not affect umbilical blood PT, PTT, or functional activity of vitamin K-dependent coagulation factors II, VII, IX, and X. However, it is associated with the reduced presence of decarboxylated prothrombin in umbilical blood at birth. There is significant improvement in umbilical blood coagulation tests as gestational age advances from 24 to 34 weeks.     

Effects of shear elasticity on sea bed scattering: numerical examples

It is known that marine sediments can support both compressional and shear waves. However, published work on scattering from irregular elastic media has not examined the influence of shear on sea bed scattering in detail. A perturbation model previously developed by the authors for joint roughness-volume scattering is used to study the effects of elasticity for three sea bed types: sedimentary rock, sand with high shear speed, and sand with "normal" shear wave speed. Both bistatic and monostatic cases are considered. For sedimentary rock it is found that shear elasticity tends to increase the importance of volume scattering and decrease the importance of roughness scattering relative to the fluid case. Shear effects are shown to be small for sands.     

Surgical anatomy for endoscopic subfascial division of perforating veins

PURPOSE: This study was undertaken to define the surgical anatomy of the medial perforating veins (PVs) of the leg and to provide information on how to gain access to all medial PVs from the superficial posterior compartment during a subfascial endoscopic procedure. METHODS: The venous anatomy of 40 limbs (from 23 cadavers) were studied. Medial PVs located between the ankle and the tibial tuberosity were dissected. None of the subjects had pathologic evidence of venous disease. Each PV's type (direct or indirect), size (< 1 mm, 1 to 2 mm, > 2 mm), location (distances from ankle [D1], and tibia [D2]), and accessibility from the superficial posterior compartment were recorded. RESULTS: Five hundred fifty-two PVs were identified (mean, 13.8; range, 7 to 22). Two hundred eighty-seven PVs (52%) directly connected the superficial with the deep systems, 228 (41%) were indirect muscle perforators, and 37 PVs (7%) were undetermined. One hundred thirty-seven PVs (25%) were > 2 mm. Sixty-three percent of PVs were accessible from the superficial posterior compartment. In the distal half of the leg, two groups of direct PVs could be identified (Cockett II: D1, 7 to 9 cm; Cockett III: D1, 10 to 12 cm). In the proximal half of the leg, paratibial direct PVs (D2 < or = 1 cm) were found clustered in three groups (D1, 18 to 22 cm; D1, 23 to 27 cm; D1, 28 to 32 cm). CONCLUSIONS: Our study confirmed the presence of the Cockett II and III PVs and three groups of proximal paratibial PVs, including the "24-cm" perforators. Two thirds of the medial direct PVs are accessible for endoscopic division from the superficial posterior compartment. To divide paratibial PVs, however, incision of the paratibial deep fascia is frequently required.     

Critical appraisal by reading for medical students--a case study from Pakistan

Doctors read literature to keep abreast of medical advances. A recommendation from the 1993 World Summit for Medical Education is that medical schools should teach medical students to critically appraise scientific reports. The Department of Community Health Sciences of Aga Khan University Medical College teaches basic research methods to medical students. This is now supplemented with "Critical Reading". Critical reading was first taught to 67 third year students between October, 1993 and May, 1994. A "validity check-list for critical readers" was introduced in a two week orientation consisting of three one-hour classroom sessions and four one-hour small group sessions. Thereafter, small groups met monthly to critique clinical epidemiological reports relevant to current organ system teaching. The students reading attitudes and critical appraisal skills were assessed through continuous assessment and a written final examination with questionnaire. All but three students passed the final examination (mean score (74%, standard deviation 12%). Sixty-four of 67 (96%) completed questionnaires. All (73% strongly) agreed that critical reading skills were essential, but only 30% strongly agreed that they had, indeed, mastered the skills. Ninety-seven percent (56% strongly) disagreed that year three was too early to start critical reading. Clinical teaching staff expressed interest in learning these skills. Students benefited from and enjoyed this first critical reading course. It strengthened ties between clinical and community health sciences teaching staff. The critical reading skills of the clinical teaching staff is being addressed in seminars to strengthen institutional research capacity.     

Protein topology of presenilin 1

Mutations in a gene encoding a multitransmembrane protein, termed presenilin 1 (PS1), are causative in the majority of early-onset cases of AD. To determine the topology of PS1, we utilized two strategies: first, we tested whether putative transmembranes are sufficient to export a protease-sensitive substrate across a lipid bilayer; and second, we examined the binding of antibodies to specific PS1 epitopes in cultured cells selectively permeabilized with the pore-forming toxin, streptolysin-O. We document that the "loop," N-terminal, and C-terminal domains of PS1 are oriented toward the cytoplasm.