The association rate constant for heme binding to globin is independent of protein structure

Rate constants for CO-heme binding to 35 different recombinant apomyoglobins and several other apoproteins were measured in an effort to understand the factors governing heme affinity and the velocity of the association reaction. Surprisingly, the rate constant for the binding of monomeric heme is approximately 1 x 10(8) M-1 s-1 regardless of the structure or overall affinity of the apoprotein for iron-porphyrin. Major differences between the proteins are reflected primarily in the rates of dissociation of the prosthetic group. Slow phases observed in the reaction of CO heme with excess apomyoglobin result from formation of nonspecific heme-protein complexes which must dissociate before heme can bind specifically in the heme pocket. Once the specific heme-globin complex is formed, the heme pocket rapidly collapses around the porphyrin, simultaneously forming the bond between the proximal His93 and the heme iron atom. The overall affinity of sperm whale apomyoglobin for hemin is approximately 1 x 10(14) M-1. Nonspecific hydrophobic interactions between the porphyrin and the apolar heme cavity account for a factor of 10(5)-10(7). Covalent bond formation between Fe3+ and His93(F8) provides an additional factor of 10(3)-10(4). Specific interactions with conserved amino acids in the heme pocket contribute the final factor of 10(3)-10(4).     

Working Group Report 2: In situ caries models, saliva, microbiology, and statistical considerations

We present a case of a single coronary artery where the right coronary artery (RCA) arose from its proximal part. This rare anomaly was detected during elective coronary angiography in a patient with suspected coronary artery disease. The single coronary artery originated from the left sinus of valsalva, giving rise to RCA proximally and distally dividing into left anterior descending (LAD), ramus intermedius and left circumflex (LCX) arteries. The anginal symptoms in this patient was attributed to a significant stenosis at the proximal LAD which was subsequently dilated by coronary angioplasty. To the best of our knowledge, this is the first reported case of angioplasty of LAD in an anomalous single coronary artery.     

Monoclonal antibodies recognizing epitopes on the extracellular face and intracellular N-terminus of the human erythrocyte anion transporter (band 3) and their application to the analysis of South East Asian ovalocytes

This report describes the production and characterization of 13 rodent monoclonal antibodies to the human erythrocyte anion transport protein AE1 (syn. band 3). Eleven antibodies (4 murine and 7 rat) recognize epitopes dependent on the integrity of the third extracellular loop of the protein. Two antibodies (1 murine and 1 rat) recognize epitopes on the N-terminal cytoplasmic domain. Quantitative binding studies using radioiodinated IgG and Fab fragments of antibodies to extracellular epitopes on AE1 ranged from 77,000 to 313,000 (IgG) and from 241,000 to 772,000 (Fab) molecules bound at saturation. The results indicate that the epitopes recognized by different antibodies vary in their accessibility and suggest that there is heterogeneity in the organization of individual AE1 molecules in the red blood cell membrane. Quantitative binding studies on South East Asian ovalocytes using several antibodies to AE1 and an anti-Wrb show a marked reduction in the number of antibody molecules bound at saturation. These results are consistent with the existence of highly cooperative interactions between transmembrane domains of AE1 in normal erythrocytes and the disruption of these interactions in the variant AE1 found in South East Asian ovalocytes.     

Clinical trial designs--made to order

At times, clinical trial design presents difficult problems for the consulting statistician. The nature of the disease under study or the inadequacies of standard methodology often suggest alternative strategies for the development of useful clinical trial designs. This paper examines two case of novel clinical trial designs that are "made-to-order" for the problem at hand. Both address disease-specific issues. The first case considers the use of adaptive allocation of treatments to patients in a trial in major depressive disorder. The second case reviews the design of a novel efficacy parameter in advanced pancreatic cancer.     

Microvascular transplantation in the salvage of lower extremity trauma in the elderly

In the cadaver of an 86 year old man the inferior segment of the azygos vein could not be found. Furthermore, a normally developed hemiazygos vein drained the right and left intercostal veins from T 10 to T 6. This vessel finally curved towards the right to reach the superior vena cava after having drained the right superior intercostal veins. The left superior intercostal vein ended in a short vessel draining into the left brachiocephalic vein. This condition may be represented in a standard chest radiograph by the so-called "aortic nipple". Agenesis of the azygos vein, suspected because of the presence of this radiological sign, should be confirmed in the living by means of computerized tomography. This can clarify the anatomy of the mediastinal vessels precisely. Embryological pattern of the azygos system accounting for its possible defects is discussed.     

A novel method for replacement of the dislodged tracheostomy tube: the nasogastric tube "guidewire" technique

The ready access to blood (plasma and formed cellular elements) makes it unusually susceptible to the deleterious effects of pollutants whose origins may be in the air. The red blood cells' hemoglobin may be rendered useless for oxygen transport by combination with carbon monoxide or conversion to methemoglobin or sulfhemoglobin. Lead and arsine can damage the erythrocytes' membranes, resulting in anemia. Metabolites of benzene and other volatile polycyclic hydrocarbons are implicated in the causation of leukemias. The extensive use of pesticides and herbicides may be associated with the development of Hodgkin's disease, non-Hodgkin's lymphoma, and aplastic anemia. The carcinogenic risks from ionizing radiation, especially for leukemia, are well known. More information is needed concerning the epidemiology of environmental factors responsible for damage to blood. Enhanced knowledge about the molecular biology of toxins' effects on the hematopoietic system and improved detection and prevention technologies are needed to answer environmentally related health questions.     

Epithelial marker genes are expressed in cultured embryonic rat lung and in vivo with similar spatial and temporal patterns

Explants of embryonic lung are often used to characterize lung growth, bronchial tree pattern, and cell differentiation. Most investigators culture lungs for 3-7 days in defined media lacking, e.g., added growth factors or hormones. If growth and differentiation are comparable to that in vivo, these cultures show considerable promise for identifying developmental regulatory molecules and target genes, and for elucidating molecular responses. We used in situ hybridization and RT-PCR to compare times and sites of expression of mRNAs of six epithelial genes in cultured and uncultured fetal rat lungs. These genes, expressed in distal lung of adult rats, are surfactant proteins (SP) A, B, and C; LAR, a receptor-type tyrosine phosphatase; Clara cell secretory protein (CC10, CCSP); and T1alpha. SP-A, SF-B, LAR, and CC10 are expressed by both Clara and Type II cells in adult animals. SP-C and T1alpha are unique markers for Type II and Type I cells, respectively. SP-C, LAR, and T1alpha are expressed before the lung is explanted (Day 13.5); SP-A, -B, and CC10 mRNAs are first detected later. The onset of expression is similar in vivo and in vitro. Although the patterns of expression differ for each mRNA, their sites of expression in culture match those in vivo relative to the bronchial tree. The explanted embryonic lung appears to be an excellent experimental model.     

Persistent thrombin generation in humans during specific thrombin inhibition with hirudin

BACKGROUND: The degree to which antithrombotic drugs suppress thrombin generation is unknown. Because hirudin, unlike antithrombin III, binds intravascular thrombin rapidly and selectively to yield a circulating inactive complex of 3- to 4-hour half-life, we used intravenous hirudin in humans to investigate the course of thrombin generation during and early after anticoagulation with this potent, direct antithrombin. METHODS AND RESULTS: Intravascular thrombin was measured with an ELISA for the thrombin-hirudin complex formed during and for 18 hours after stopping a 6-hour infusion of hirudin at 0.1, 0.2, and 0.3 mg.kg-1.h-1 in three groups of six patients each. With free hirudin in 20- to 10,000-fold molar excess of thrombin and peak activated partial thromboplastin times of 2.3 to 3.0 times baseline, mean plasma thrombin-hirudin complex increased from 794 +/- 85 pg/mL (mean +/- SEM) 15 minutes after the start of the infusion to 1617 +/- 151 pg/mL at 6 hours of infusion to 2667 +/- 654 pg/mL at 24 hours. During the 24-hour observation period, plasma concentration of fragment 1.2 (the peptide released during conversion of prothrombin to thrombin) never fell below baseline but rather increased transiently during the hirudin infusion. Plasma concentrations of thrombin-antithrombin III complex (in ng/mL) decreased from 4.34 +/- 0.40 at baseline to 1.64 +/- 0.13 at 6 hours (P < .001) and gradually increased after stopping the infusion to 5.7 +/- 0.87 at 24 hours (nonsignificant compared with baseline). CONCLUSIONS: Measurement of thrombin-hirudin complex may be used as a marker of thrombin generation in humans. Persistent accumulation of thrombin-hirudin complex and generation of fragment 1.2 during and after completion of potent anticoagulation with hirudin suggest thrombin generation is not blocked by high-affinity thrombin inhibition. The persistent formation of thrombin during declining plasma levels of hirudin may contribute to the pathogenesis of rethrombosis early after antithrombin therapy or during inadequate anticoagulation.     

Epilepsy in very preterm infants: neonatal cranial ultrasound reveals a high-risk subcategory

Osteoporotic fractures, and in particular, hip fractures result in significant morbidity and mortality. Low bone mass is the main risk factor of enhanced bone fragility, resulting in an increased risk for hip fracture. Bone density of osteoporotic women with and without hip fractures show a considerable overlap. Therefore, other bone-independent factors also play an important role for the development of hip- and other osteoporotic fractures. One other important factor is falling. In 90% of hip fractures falling was involved [10-15], but only 5% or less of these falls resulted in a subsequent fracture. The view that adequate exercise is beneficial for skeletal health of children and for prevention and treatment of osteoporosis in adults is supported primarily by two lines of evidence: longitudinal and cross-sectional trials in children and young adult athletes showing a significant increase of muscle- and bone mass after strenuous (children) or chronic exercise (athletes) as compared to normally active (children) or sedentary control subjects. What are the potential benefits and limits of specific exercise programs with respect to bone mass, prevention of falls and fractures? In this review these questions are discussed and a specific exercise program in osteoporotic patients with fractures is delineated.