The kinetics of the reversible inhibition of heart lactate dehydrogenase through the formation of the enzyme–oxidized nicotinamide–adenine dinucleotide–pyruvate compound

The inhibition of lactate dehydrogenase at high pyruvate concentration was studied in three ways. First, a rapid decrease in the rate of the enzyme reaction was observed; secondly, the rate of formation of a pyruvate-NAD(+) compound was followed by the change in E(325); thirdly, the rate of quenching of the protein fluorescence was measured. The data obtained at pH6.0 at different temperatures and ionic strengths as functions of pyruvate, NAD(+) and enzyme concentrations show that the extent of inhibition can be correlated with the reversible formation of a compound between pyruvate and enzyme-bound NAD(+). It is suggested that the detailed kinetic analysis of the formation of this abortive ternary compound will give pertinent information about properties of the enzyme-NAD(+) compound involved in the normal catalytic process.     

Maternal body mass index and risk of testicular cancer in male offspring: A systematic review and meta-analysis

Objectives: To date a number of studies have examined the association between maternal weight and testicular cancer risk although results have been largely inconsistent. This systematic review and meta-analysis investigated the nature of this association. Methods: Search strategies were conducted in Ovid Medline (1950–2009), Embase (1980–2009), Web of Science (1970–2009), and CINAHL (1937–2009) using keywords for maternal weight (BMI) and testicular cancer. Results: The literature search produced 1689 hits from which 63 papers were extracted. Only 7 studies met the pre-defined criteria. Random effects meta-analyses were conducted. The combined unadjusted OR (95% CI) of testicular cancer in the highest reported category of maternal BMI compared with the moderate maternal BMI was 0.82 (0.65–1.02). The Cochran's Q P value was 0.82 and the corresponding I² was 0%, both indicating very little variability among studies. The combined unadjusted OR (95% CI) for testicular cancer risk in the lowest reported category of maternal BMI compared to a moderate maternal BMI category was 0.88 (0.65–1.20). The Cochran's Q P value was 0.05 and the corresponding I² was 54%, indicating evidence of statistical heterogeneity. The combined unadjusted OR (95% CI) of testicular cancer risk per unit increase in maternal BMI was 1.01 (0.97–1.06). The Cochran's Q test had a P value of 0.05 and the corresponding I² was 55% indicating evidence of statistical heterogeneity. Conclusion: This meta-analysis, which included a small number of studies, showed that a higher maternal weight does not increase the risk of testicular cancer in male offspring. Though an inverse association between high maternal BMI and testicular cancer risk was detected, it was not statistically significant. Further primary studies with adjustment for appropriate confounders are required.     

CheZ phosphatase localizes to chemoreceptor patches via CheA-short

We have investigated the conditions required for polar localization of the CheZ phosphatase by using a CheZ-green fluorescent protein fusion protein that, when expressed from a single gene in the chromosome, restored chemotaxis to a DeltacheZ strain. Localization was observed in wild-type, DeltacheZ, DeltacheYZ, and DeltacheRB cells but not in cells with cheA, cheW, or all chemoreceptor genes except aer deleted. Cells making only CheA-short (CheA(S)) or CheA lacking the P2 domain also retained normal localization, whereas cells producing only CheA-long or CheA missing the P1 and P2 domains did not. We conclude that CheZ localization requires the truncated C-terminal portion of the P1 domain present in CheA(S). Missense mutations targeting residues 83 through 120 of CheZ also abolished localization. Two of these mutations do not disrupt chemotaxis, indicating that they specifically prevent interaction with CheA(S) while leaving other activities of CheZ intact.     

A genomewide scan for loci involved in attention-deficit/hyperactivity disorder

Attention deficit/hyperactivity disorder (ADHD) is a common heritable disorder with a childhood onset. Molecular genetic studies of ADHD have previously focused on examining the roles of specific candidate genes, primarily those involved in dopaminergic pathways. We have performed the first systematic genomewide linkage scan for loci influencing ADHD in 126 affected sib pairs, using a approximately 10-cM grid of microsatellite markers. Allele-sharing linkage methods enabled us to exclude any loci with a lambda(s) of > or =3 from 96% of the genome and those with a lambda(s) of > or =2.5 from 91%, indicating that there is unlikely to be a major gene involved in ADHD susceptibility in our sample. Under a strict diagnostic scheme we could exclude all screened regions of the X chromosome for a locus-specific lambda(s) of >/=2 in brother-brother pairs, demonstrating that the excess of affected males with ADHD is probably not attributable to a major X-linked effect. Qualitative trait maximum LOD score analyses pointed to a number of chromosomal sites that may contain genetic risk factors of moderate effect. None exceeded genomewide significance thresholds, but LOD scores were >1.5 for regions on 5p12, 10q26, 12q23, and 16p13. Quantitative-trait analysis of ADHD symptom counts implicated a region on 12p13 (maximum LOD 2.6) that also yielded a LOD >1 when qualitative methods were used. A survey of regions containing 36 genes that have been proposed as candidates for ADHD indicated that 29 of these genes, including DRD4 and DAT1, could be excluded for a lambda(s) of 2. Only three of the candidates-DRD5, 5HTT, and CALCYON-coincided with sites of positive linkage identified by our screen. Two of the regions highlighted in the present study, 2q24 and 16p13, coincided with the top linkage peaks reported by a recent genome-scan study of autistic sib pairs.     

Quantitative assessment of the quality of medical care provided in five family practices before and after attachment of a family practice nurse.

The standards of patient care were maintained in five urban medical practices after the introduction of family practice nurses. Evaluations were achieved before and after their appointment by the indicator condition method. Minimal explicit criteria for the management of patients with 12 indicator conditions and by the use of 14 drugs were approved by an ad hoc peer group of community physicians. These cirteria were applied to the five practices by the use of a single-blind design and the abstraction of unaltered medical records. A standardized score for each practic e permitted comparison of scores for the management of indicator conditions and for the clinical use of drugs before and after attachment of the family practice nurses. For each of the indicator conditions and the drugs assessed in the five practices similar levels of adequacy were observed in the two study periods. These explicit (objective) audit resutls agreed with the implicit (subjective) assessments of the family practice nurses by their physician colleagues.     

The implication of de novo 21-hydroxylase mutation in clinical and prenatal molecular diagnoses

We studied 37 unrelated families with a history of 21-hydroxylase deficiency (CYP21D) for eight common mutations and gene deletions in the 21-hydroxylase (CYP21) gene. We found de novo mutations in the CYP21 gene in two CYP21D patients. Analysis for eight common mutations in the 21-hydroxylase gene as well as large gene deletions was accomplished using polymerase chain reaction (PCR) followed by amplified created restriction site (ACRS) or restriction fragment length polymorphism (RFLP) and Southern blot followed by hybridization to a CYP21-specific probe. Linkage analysis was performed using microsatellite markers flanking the CYP21 gene. Ten short tandem repeat (STR) markers were used to confirm parentage in the two de novo mutation cases. In two prenatal diagnosis cases, an intron 2-13A/C>G mutation was identified in the proband, but not in the fetus, although the proband and fetus had identical linkage markers. Subsequently, the mutation was confirmed to be absent in the parents' genome and misparentage was ruled out. Our findings are consistent with previous studies showing a de novo mutation frequency of approximately 1.0-1.5% in the CYP21 gene. This new mutation rate is high relative to the rate of approximately one in one million for other autosomal recessive disorders. Thus, the de novo mutation rate in the CYP21 gene is not negligible. It must be considered and discussed in prenatal diagnosis and genetic counseling for this relatively common inherited disorder.     

Molecular mapping of thrombin-receptor interactions

In addition to its procoagulant and anticoagulant roles in the blood coagulation cascade, thrombin works as a signaling molecule when it interacts with the G-protein coupled receptors PAR1, PAR3, and PAR4. We have mapped the thrombin epitopes responsible for these interactions using enzymatic assays and Ala scanning mutagenesis. The epitopes overlap considerably, and are almost identical to those of fibrinogen and fibrin, but a few unanticipated differences are uncovered that help explain the higher (90-fold) specificity of PAR1 relative to PAR3 and PAR4. The most critical residues for the interaction with the PARs are located around the active site where mutations affect recognition in the order PAR4 > PAR3 > PAR1. Other important residues for PAR binding cluster in a small area of exosite I where mutations affect recognition in the order PAR1 > PAR3 > PAR4. Owing to this hierarchy of effects, the mutation W215A selectively compromises PAR4 cleavage, whereas the mutation R67A abrogates the higher specificity of PAR1 relative to PAR3 and PAR4. 3D models of thrombin complexed with PAR1, PAR3, and PAR4 are constructed and account for the perturbations documented by the mutagenesis studies.