Molecular characterization of accessions of Cratylia argentea (Camaratuba) using ISSR markers.

Cratylia argentea (Desv.) Kuntze (Fabaceae) is a drought-tolerant, perennial legume found primarily in Brazil, Bolivia, and Peru. The shrub is well adapted to acid soils and exhibits high productivity and nutritional value, characteristics that would favor its use as a dry season animal forage supplement in semiarid regions. In plant improvement programs, the production of elite hybrids with superior traits is generally achieved by crossing parents that exhibit the highest level of genetic divergence. Therefore, the aim of the present study was to assess genetic diversity among 13 accessions of C. argentea from the same population maintained in the active germplasm bank of Embrapa Meio-Norte using inter-simple sequence repeat (ISSR) markers. Genetic similarities between C. argentea accessions were estimated from Jaccard coefficients, and a dendrogram was constructed using the unweighted pair group method with arithmetic average (UPGMA). The set of 15 primers selected for ISSR analysis generated a total of 313 loci of which 79.23% were polymorphic. The mean number of bands per primer was 20.87, and the amplicons ranged from 280 to 3000 bp in size. Primers UBC834 and UBC827 generated the largest number of polymorphic loci and exhibited 90.91 and 100% polymorphism, respectively. The coefficients of genetic similarity among accessions varied between 0.49 and 0.73. UPGMA cluster analysis allowed the identification of four genotypic groups and demonstrated the existence of considerable variability within the collection. Potential progenitors were selected that would offer good possibilities of obtaining unusual and favorable combinations of genes in a plant breeding program.

Comparison of methods to isolate DNA from Caesalpinia ferrea.

Molecular markers are important for characterizing the genetic diversity of plants and can provide the basis for strategies to protect and conserve endangered populations. However, numerous molecular techniques are used, requiring an evaluation of fast and efficient methods to extract DNA. Since molecular studies of Caesalpinia ferrea are rare, it is important to develop and/or adapt a DNA extraction protocol that produces quality DNA samples to enable the design of strategies for the conservation of this threatened species. This study aimed to compare five methods for DNA extraction and to determine the most efficient protocol for C. ferrea. Sufficient genomic DNA was obtained from the leaves of C. ferrea using all the tested protocols to perform techniques involving molecular markers. Two protocols based on the detergent cetyl trimethyl ammonium bromide, as well as a commercial kit, yielded high concentrations of pure DNA. However, when polymerase chain reaction amplifications were performed, DNA was only successfully amplified from extractions performed with the commercial kit, which produced sufficient genomic DNA of good quality from the leaves of C. ferrea to perform techniques involving molecular markers.

Effect of heparin loading during congenital heart operation on thrombin generation and blood loss.

BACKGROUND: The heparin protocols used during cardiopulmonary bypass (CPB) in children undergoing surgical repair for congenital heart disease are extrapolated from adult data. Studies are needed that assess the optimal heparin dosing in these children, whose heparin clearance is increased compared with that in adults. METHODS: We assessed the effects of two commonly used doses of heparin in the prime solution at the start of CPB operation on plasma heparin levels, on thrombin production (thrombin-antithrombin III complexes, prothrombin fragment 1 + 2, D-dimer, and antithrombin III), and on the risk of hemorrhage. Before CPB, 48 children with congenital heart disease received heparin intravenously in a loading dose of 300 U/kg, followed by either 1 U/mL of heparin in the prime (low-dose group: 22 patients-acyanotic, 9; cyanotic, 13) or 3 U/mL of heparin in the prime (group: high-dose, 26 patients-acyanotic, 15; cyanotic, 11). RESULTS: In all patients, CPB resulted in the generation of thrombin. The duration of CPB was a significant covariate factor for heparin levels (p = 0.002), thrombin production (p < 0.001), and postoperative blood loss (p < 0.001). In the patients in the high-dose group, the total heparin dose and the plasma heparin levels were higher (p = 0.0005 and 0.005, respectively) and the D-dimer levels tended to be lower (p = 0.06). The postoperative blood loss was higher in the cyanotic patients (p = 0.02; both high-dose and low-dose groups), with 2 cyanotic patients (1 in low-dose group, 1 in high-dose group) requiring reoperation, one of whom subsequently died. The increased heparin dose had no significant effect on the rate or volume of postoperative blood loss. CONCLUSIONS: Increasing the heparin dose in the prime solution from 1 to 3 U/mL increased the plasma heparin levels and showed a trend toward reducing the postoperative laboratory values indicative of fibrinolysis. Thrombin generation during CPB and the incidence of postoperative hemorrhage were not significantly altered. Larger randomized trials are needed to determine the optimal heparin-dosing regimen in patients with congenital heart disease.

Cardiorespiratory status after treatment for acute lymphoblastic leukemia.

The use of certain chemotherapeutic agents is associated with dose-related cardiotoxicity and, potentially, with restrictive lung disease. Therefore, we assessed the cardiopulmonary status and exercise capacity of 19 patients (pts; 9M:10F) 1.1 to 7.1 years (mean 4.6 +/- 1.5 years) after successful treatment of acute lymphoblastic leukemia (ALL) with Dana Farber Cancer Institute protocols. As body mass and nutritional status may influence exercise capacity, we also evaluated their anthropometric status and the plasma levels of rapid turnover proteins. Seven pts designated as "standard risk for relapse" (SR) had received low cumulative doses of doxorubicin (50 +/- 21 mg/m2), while twelve pts at "high or very high risk for relapse" (HR/VHR) had received higher doses (349 +/- 16 mg/m2). The evaluations included a questionnaire, anthropometric assessments, echocardiography, pulmonary function studies, exercise testing, and nutritional assays. Patients' data were compared with published normative data or with control values from our laboratories. In addition, we compared SR pt data with HR/VHR pt data. No pt had overt symptoms or signs of cardiorespiratory compromise. The pts had a higher percent of body fat than age-matched healthy controls (29.7 +/- 7.9% vs. 20 +/- 6%; P < 0.001). On echocardiography, cardiac systolic function was within normal limits in all. However, HR/VHR pts had lower left ventricular (LV) shortening fractions than SR pts (P < 0.05). LV filling velocity, indicative of diastolic function (the E/A ratio), was normal in most pts. Pulmonary function studies were normal. Exercise capacity was below predicted in most cases but heart rates at peak exercise and leg muscle function were within normal limits, suggesting a deconditioned state. Plasma levels of rapid turnover proteins were also normal. Despite lack of overt morbidity in our pt population, subtle abnormalities persist in cardiac function while pulmonary function is normal. Longitudinal studies will identify if further abnormalities or overt morbidity develop. In later years, continuing obesity and a sedentary state may contribute to clinically relevant heart disease.

Thrombin regulation in congenital heart disease after cardiopulmonary bypass operations.

Children with cyanotic congenital heart disease who undergo operation with cardiopulmonary bypass are at increased risk of thromboembolic or hemorrhagic complications, or both. Regulation of thrombin, a key enzyme in coagulation, is essential in preventing these complications. We therefore examined the in vitro capacity of plasma from 15 children with cyanotic congenital heart disease to generate thrombin and to inhibit 125I-thrombin before and after cardiopulmonary bypass. We also assessed whether thrombin had been generated in vivo by assaying levels of fibrinogen, thrombin-antithrombin III complexes, and D-dimer. Plasma levels of the thrombin inhibitors, antithrombin III, alpha-2-macroglobulin, and heparin cofactor II were also measured. Thrombin regulation was normal before operation. After cardiopulmonary bypass, the in vitro capacity to generate thrombin decreased by 50%, and this was primarily a result of hemodilution (31%). Similar postoperative decreases were noted in the levels of antithrombin III, heparin cofactor II, and alpha-2-macroglobulin (26% to 45%). However, the total in vitro plasma thrombin inhibitory capacity decreased by only 13%. Levels of thrombin-antithrombin III and D-dimer increased after operation, indicating that thrombin had been generated and inhibited in vivo. Clinically, there were no thromboembolic complications although six patients required replacement therapy for excessive small-vessel bleeding. In conclusion, thrombin regulation is significantly altered after cardiopulmonary bypass. Although thrombin is generated in vivo, the total residual capacity to do so is impaired because of hemodilution. Despite a concomitant decrease in thrombin inhibitor levels, the total residual in vitro capacity of plasma to inhibit thrombin is relatively spared. This suggests that after cardiopulmonary bypass the risk of hemorrhagic complications after an additional hemostatic challenge is relatively greater than the risk of thrombotic complications. This might be reflected in the predominance of hemorrhagic complications in our patients.

Abnormalities in von Willebrand factor and antithrombin III after cardiopulmonary bypass operations for congenital heart disease.

In patients with congenital heart disease two poorly understood postoperative complications are pulmonary hypertensive crises after repair of large atrioventricular or ventricular septal defects and right atrial and pulmonary thrombi after the Fontan operation. In this study we assessed whether cardiopulmonary bypass in these patients is associated with the release of agents that might induce platelet aggregation and vasoconstriction, such as biologically active von Willebrand factor and platelet-activating factor. In addition, we measured levels of anticoagulants such as antithrombin III and proteins C and S. Three groups of patients with congenital heart disease undergoing cardiopulmonary bypass were monitored through the perioperative period for secundum atrial septal defects, large atrioventricular or ventricular septal defects, and tricuspid atresia or univentricular heart (Fontan candidates). Control values were obtained from age-matched patients; patients requiring major noncardiac operations and those with cardiac disease not requiring cardiopulmonary bypass were also studied. After cardiopulmonary bypass in all three groups biologic activity of von Willebrand factor increased markedly in the immediate and early postoperative periods compared with preoperative values, whereas antithrombin III values were decreased. Platelet-activating factor was detected in only two patients with congenital heart disease, both in the early postoperative period. In contrast, patients who did not have cardiopulmonary bypass did not show these abnormalities. All measured parameters normalized at late follow-up (6 to 18 months after operation). Although cardiopulmonary bypass in these patients resulted in increased von Willebrand factor activity and decreased antithrombin III, changes that may predispose the patient to platelet aggregation and thrombus formation, absolute values in individual patients alone were not predictive of pulmonary hypertensive crises or detectable thrombi. This suggests that these hematologic abnormalities may contribute to but are not by themselves a cause of morbidity in the early postoperative period. Moreover, the increased von Willebrand factor biologic activity seen postoperatively in patients with congenital heart disease suggests that use of synthetic vasopressin may be ineffective and potentially detrimental.

Effect of ambient oxygen changes on platelet activating factor production by fetal ovine endothelial cells.

To determine whether platelet activating factor (PAF) plays a role in the responses seen in the fetal and transitional circulations, we assessed endogenous release of PAF in cultured fetal ovine endothelial cells from the pulmonary artery (PA), ductus arteriosus (DA) and aorta (Ao) under basal conditions and following exposure to hypoxia or hyperoxia. The cells were prelabeled with [3H] acetate and subsequently exposed to different ambient oxygen concentrations, i.e., 95% O2 or 95% N2, balance CO2, using calcium ionophore as a positive control. The effect of indomethacin on DA endothelial PAF production following stimulation with ionophore was also established. Synthesis of [3H] PAF was measured by counts comigrating on TLC with unlabeled PAF. We found that PAF production by fetal ovine PA, Ao and DA cells was similar and unaffected by hypoxia or hyperoxia. Exposure of ionophore stimulated DA cells to indomethacin was, however, associated with a decrease in PAF production (p less than 0.05). We speculate that in vitro alterations in ambient O2 concentration do not influence fetal ovine endothelial PAF production but indomethacin may decrease PAF production in the DA.

Persistence of atrioventricular valve regurgitation and electrocardiographic abnormalities following transient myocardial ischemia of the newborn.

To determine the sequelae of transient myocardial ischemia (TMI) in term infants, we reviewed clinical and investigative data in 59 infants (37 male, 22 female) with structurally normal hearts admitted over the 2-year period of 1983-1985. Twenty-three were diagnosed prior to admission as cases of birth asphyxia (5-min Apgar score less than 6), and 36 had signs of persistent fetal circulation with electrocardiographic (ECG) changes of ischemia greater than 24 h after birth. Murmurs of atrioventricular valve regurgitation (AVVR), detected in 28 patients, were confirmed in 23 of the 24 patients investigated. The murmurs resolved over a 2-day to 6-month period (median 6 days). In three patients, AVVR, left ventricular dyskinesia, and ECG anomalies persisted for 2 months (until death), 4 months, and 48 months. Initial ECGs were abnormal in 57 patients, and (of those reviewed) 60% returned to normal over a 6-day to 7-month period (median 2 months). Residual ECG anomalies included second-degree AV block and persistent ST-T wave changes. Ten patients died from noncardiac causes. Neither the presence nor resolution of AVVR correlated significantly with the severity of birth asphyxia using the Apgar score, nor with the severity of the ischemic changes on the ECG. Although the cardiovascular sequelae of myocardial ischemia are usually transient, the data should prompt the need for careful review after the initial admission.

A rare cause of spinal cord compression in childhood from intraspinal mesenchymal chondrosarcoma. A report of two cases and review of the literature.

Two children presented with acute spinal cord compression by primary and metastatic intraspinal mesenchymal chondrosarcoma, a rare pediatric malignancy. Patients with the primary intraspinal tumor usually present early and often respond well to combined surgery, irradiation and chemotherapy. Patients with intraspinal metastases present late in the course of their disease and their prognoses are poor.