Purification and characterization of branched chain α-keto acid dehydrogenase complex of bovine kidney

A branched chain alpha-keto acid dehydrogenase-dihydrolipoyl transacylase complex was purified to apparent homogeneity from bovine kidney mitochondria. As usually isolated, the complex (s(20,w) = 40 S) contained little, if any, dihydrolipoyl dehydrogenase. When saturated with the latter enzyme the complex had a specific activity of about 12 mumol of alpha-ketoisovalerate oxidized per min per mg of protein at 30 degrees with NAD(+) as electron acceptor. In addition to alpha-ketoisovalerate, the complex also oxidized alpha-ketoisocaproate, alpha-keto-beta-methylvalerate, alpha-ketobutyrate, and pyruvate. The ratios of the specific activities were 2.0:1.5:1.0:1.0:0.4, and the apparent K(m) values were 40, 50, 37, 56, and 1000 muM. The complex was separated into its component enzymes. The branched chain alpha-keto acid dehydrogenase (6 S) consists of two different subunits with estimated molecular weights of 46,000 and 35,000. The dihydrolipoyl transacylase (20 S) contains apparently identical subunits of molecular weight about 52,000. In the electron microscope, the transacylase has the appearance of a cube, and the molecules of branched chain alpha-keto acid dehydrogenase appear to be distributed on the surface of the cube. In contrast to the pyruvate dehydrogenase complex of bovine kidney, the branched chain alpha-keto acid dehydrogenase complex apparently is not regulated by phosphorylation-dephosphorylation. Its activity, however, is subject to modulation by end-product inhibition.     

Analysis of Iranian patients allowed the identification of the first truncating mutation in the fibrinogen Bbeta-chain gene causing afibrinogenemia

BACKGROUND AND OBJECTIVES: Congenital afibrinogenemia is a rare coagulation disorder whose molecular basis is still poorly characterized. Most mutations have been identified in the fibrinogen Aalpha- and gamma-chain genes, whereas only two missense mutations have been reported in the Bbeta-chain gene. The aim of this work was to widen knowledge about the mutational spectrum of this disease by analyzing the molecular bases of congenital afibrinogenemia in three unrelated Iranian patients. DESIGN AND METHODS: All patients showed unmeasurable levels of clottable fibrinogen in plasma. Mutational screening was performed by sequencing the whole coding region, including exon-intron boundaries and part of the promoter region of the three fibrinogen genes. RESULTS: Sequencing in one patient revealed the presence of a novel nonsense mutation (3282C-->T) in exon 2 of the fibrinogen Bbeta-chain gene, causing a severe truncation of the corresponding polypeptide (R17X). In the remaining probands, two already known small deletions (4209delA and 4220delT), both located in exon 5 of the fibrinogen Aalpha-chain gene, were identified, and their effect at the protein level explored by computer-assisted analysis. INTERPRETATION AND CONCLUSIONS: The identification of the first truncating mutation in the fibrinogen Bbeta-chain gene confirms the involvement of all three fibrinogen genes in the pathogenesis of congenital afibrinogenemia and widens the mutational spectrum of the disease. This knowledge is clinically essential in order to carry out prenatal diagnosis in families at risk.     

Cardiometabolic differences in children born after in vitro fertilization: follow-up study

CONTEXT: Increasing evidence suggests that adverse conditions during early prenatal life are associated with cardiometabolic dysfunction in postnatal life. In vitro fertilization (IVF) conception may be an early prenatal life event with long-term health consequences. OBJECTIVE: Our objective was to investigate several cardiometabolic measures in 8- to 18-yr-old IVF singletons and spontaneously conceived controls born from subfertile parents. DESIGN AND SETTING: This follow-up study was conducted at the VU University Medical Center, Amsterdam, The Netherlands. PARTICIPANTS: Blood pressure was examined in 225 IVF-conceived children and 225 age- and gender-matched spontaneously conceived control children. Several indicators of insulin resistance were studied in a pubertal subpopulation (131 IVF children and 131 controls). MAIN OUTCOME MEASURES: Blood pressure, fasting glucose, and fasting insulin were determined. RESULTS: Systolic and diastolic blood pressure levels were higher in IVF children than controls (109 +/- 11 vs. 105 +/- 10 mm Hg, P < 0.001; and 61 +/- 7 vs. 59 +/- 7 mm Hg, P < 0.001, respectively). Children born after IVF were also more likely to be in the highest systolic and diastolic blood pressure quartiles (odds ratio = 2.1, 95% confidence interval 1.4, 3.3; odds ratio = 1.9, 95% confidence interval 1.2, 3.0, respectively). Furthermore, higher fasting glucose levels were observed in pubertal IVF children (5.0 +/- 0.4 vs. 4.8 +/- 0.4 mmol/liter in controls; P = 0.005). Blood pressure and fasting glucose differences could not be explained by current body size, birth weight, and other early life factors or by parental characteristics, including subfertility cause. CONCLUSIONS: These findings highlight the importance of continued cardiometabolic monitoring of IVF-conceived children and might contribute to current knowledge about periconceptional influences and their consequences in later life.     

The P303T mutation in the human factor VII (FVII) gene alters the conformational state of the enzyme and causes a severe functional deficiency

We report the results of in vitro expression and biochemical characterization of the naturally occurring type II mutation Pro303Thr (P303T) in the factor VII (FVII) gene. Recombinant activated mutated FVII (FVIIa303T), compared with the activated wild-type FVII (FVIIaWT), showed reduced amidase activity toward synthetic substrates, especially when the observed reduced binding affinity for human soluble tissue factor (TF) (K(d) from 4.4 nmol/l for FVIIaWT to 17.3 nmol/l for FVIIa303T) was overcome by a fully saturating TF concentration. Likewise, factor X (FX) hydrolysis by FVIIa303T showed a reduced activity in the absence (and more severely in the presence) of TF (k(cat)/K(m) from 2.3 x 10(7)/mol/l s for FVIIaWT to 8.7 x 10(5)/mol/l s for FVIIa303T). These results showed that the mutant FVIIa is more shifted toward a zymogen-like form compared to FVIIaWT, suggesting that P303 facilitates the conformational transitions that stabilize the active form of FVIIa. The alteration of these allosteric equilibria is especially evident in the presence of TF, which was unable to shift the equilibrium toward a fully active FVIIa form. Additional experiments showed that both TF-catalysed FVII303T autoactivation and FVII303T activation by activated FX in the presence of TF were severely impaired, mainly because of an increase of the K(m) value. Altogether, these defects may explain the severe bleeding symptoms in a patient carrying the FVIIP303T mutation.     

Does comorbidity explain trends in prescribing of newer antihypertensive agents?

OBJECTIVE: Concerns exist about heavily prescribing of new drugs when the evidence on hard outcomes is still limited. This has been the case for the newer classes of antihypertensives, especially in hypertensive patients without additional comorbidity. The association between comorbidity and trends in prescribing of angiotensin-converting enzyme inhibitors (ACE-I) and angiotensin II receptor blockers (ARBs) was examined for the period 1996-2000. DESIGN AND METHODS: Data were obtained from the Integrated Primary Care Information database, which contains medical records from more than 100 general practitioners in the Netherlands. Prevalent drug use in hypertensive patients was determined per calendar year. As initial treatment, the first antihypertensive drug prescribed within 1 year after diagnosis of hypertension was considered. Logistic regression was used to estimate the likelihood of receiving either ACE-I or ARBs. RESULTS: The overall prevalent ACE-I use remained stable (31%), but it increased from 33 to 41% in hypertensive patients with diabetes, heart failure, proteinuria and/or renal insufficiency. ARB use increased significantly from 2 to 12%; this trend did not differ between patients with or without specific comorbidities. Initial ACE-I use slightly decreased (from 29% to 24%), whereas initial ARB use significantly increased (from 4% to 12%). ACE-I were more likely to be the first treatment in patients with diabetes [odds ratio (OR)=3.9; 95% confidence interval (CI) 3.2-4.9] or hypercholesterolemia (OR=1.4; 95% CI 1.1-1.8). ARBs were more likely to be the initial treatment in patients with asthma/chronic obstructive pulmonary disease (OR=1.6; 1.2-2.3), diabetes (OR=2.1; 1.5-2.9) or hypercholesterolemia (OR=1.7; 1.2-2.4). CONCLUSIONS: The increased use of ACE-I is mostly restricted to hypertensive patients with comorbidities for which their use has been recommended. Trends in prescribing of ARBs are not related to relevant comorbidities.     

The relevance of heart failure severity for treatment with evidence-based pharmacotherapy in general practice

AIMS: Internationally, research indicates that pharmacotherapy for chronic heart failure (CHF) is sub-optimal. Traditionally, assessment of drug use in heart failure has focused on the use of individual agents irrespective of CHF severity. This study investigates drug use for CHF patients in general practice with respect to the available evidence, incorporating both disease severity and the use of combination drug regimes. METHODS AND RESULTS: A cross-sectional survey of 769 Dutch CHF patients was performed as part of IMPROVEMENT of HF study. For each New York Heart Association severity classification the minimum treatment appropriate for the heart failure severity according to the scientific evidence available at the time of the study (1999) was defined. The proportion of patients treated with each drug increased with increasing severity, with the exception of the beta-blockers. Patients with less severe heart failure were approximately four to eight times more likely to receive evidence-based treatment than those with more severe heart failure. DISCUSSION: To assess pharmacological treatment of heart failure, in relation to the available evidence, it is important to take severity into account. While the number of drugs prescribed increased with increasing severity, the use of evidence-based regimes was lower in patients with more severe heart failure.     

The enigma of quality of life in patients with heart failure

Current treatment goals in heart failure (HF) aim to improve both survival and quality of life (QoL) of patients. In this brief communication, we reviewed randomized controlled trials that assessed the impact of pharmacological treatment on QoL, and we discussed some methodological limitations of QoL assessment in HF. Studies that assessed QoL with a disease-specific questionnaire were included. We found that at present there is a paradox in HF treatment. Life prolonging therapies, such as angiotensin-converting-enzyme-inhibitors, and angiotensin receptor blockers improve modestly or only delay the progressive worsening of QoL in HF. Treatment with beta blockers does not affect QoL in any way. However, this neutral effect of beta blockers may also be due to some methodological limitations, such as the small number of patients included in beta blocker trials or the short duration of follow-up. Disease-specific questionnaires may also have some limitations, e.g. are not sensitive enough to detect small changes in QoL. On the other hand, therapies that significantly improve QoL in HF (e.g. inotropic agents) do not seem beneficial in relation to survival. We conclude that QoL in HF remains an open field, in which new therapies but also clarification of methodology is required. In the mean time, the use of life prolonging therapies appears as a safe measure to modestly improve or maintain QoL.     

The Impact of the Good Behavior Game,a Universal Classroom-Based Preventive Intervention in First and Second Grades,on High-Risk Sexual Behaviors and Drug Abuse and Dependence Disorders into Young Adulthood

The Good Behavior Game (GBG), a method of teacher classroom behavior management, was tested in first- and second-grade classrooms in 19 Baltimore City Public Schools beginning in the 1985–1986 school year. The intervention was directed at the classroom as a whole to socialize children to the student role and reduce aggressive, disruptive behaviors, confirmed antecedents of a profile of externalizing problem outcomes. This article reports on the GBG impact on the courses and interrelationships among aggressive, disruptive behavior through middle school, risky sexual behaviors, and drug abuse and dependence disorders through ages 19–21. In five poor to lower-middle class, mainly African American urban areas, classrooms within matched schools were assigned randomly to either the GBG intervention or the control condition. Balanced assignment of children to classrooms was made, and teachers were randomly assigned to intervention or control. Analyses involved multilevel growth mixture modeling. By young adulthood, significant GBG impact was found in terms of reduced high-risk sexual behaviors and drug abuse and dependence disorders among males who in first grade and through middle school were more aggressive, disruptive. A replication with the next cohort of first-grade children with the same teachers occurred during the following school year, but with minimal teacher mentoring and monitoring. Findings were not significant but generally in the predicted direction. A universal classroom-based prevention intervention in first- and second-grade classrooms can reduce drug abuse and dependence disorders and risky sexual behaviors.