Engineering lysine reactivity as a conformational sensor in the Dictyostelium myosin II motor domain

Lys84 of skeletal muscle myosin located at the interface between the motor and neck domains has long been utilized as a useful chemical probe sensing motor domain conformational changes and tilting of the lever arm. Here we report the first site-directed mutagenesis study on this side chain and its immediate chemical environment. We made Dictyostelium myosin II motor domain constructs in which Lys84 was replaced by either a methionine or a glutamic acid residue and another mutant containing an Arg704Glu substitution. By following trinitrophenylation of the mutant constructs, we first unambiguously identify Lys84 as the reactive lysine in Dictyostelium myosin. Analysis of the reaction profiles also reveals that the Lys84-Arg704 interaction at the interface of two subdomains of the myosin head has a significant effect on Lys84 reactivity, but it is not the only determinant of this property. Our findings imply that the nucleotide sensitivity of the trinitrophenylation reaction is a general feature of conventional myosins that reflects similar changes in the conformational dynamics of the different orthologs during the ATPase cycle.     

Conventional radiation therapy in the management of arteriovenous malformations of the central nervous system

Between 1967 and 1981, five patients were treated for vascular malformation of the central nervous system using conventional radiotherapy at Stanford University Medical Center. Two patients had arteriovenous malformations (AVM's) of the spinal cord, two patients had intracranial AVM's, and one patient had a hemangio-epithelioma of the occiput. All patients demonstrated clinical improvement after high dose radiation therapy with follow-up from 5 to 20 years. There have been no complications directly attributable to radiation therapy.     

ALK-positive anaplastic large T-cell lymphoma preceded by Epstein-Barr virus infection complicated by development of an aorto-esophageal fistula

We report an unusual case of aggressive ALK-positive anaplastic large cell lymphoma with widespread mediastinal involvement immediately preceded by an acute Epstein-Barr virus (EBV) infection. Following initiation of chemotherapy and radiological evidence of significant tumor regression, the patient suffered a fatal massive upper gastrointestinal hemorrhage from an aorto-esophageal fistula. The relevant literature relating to EBV in the pathogenesis of ALK-lymphomas and literature relating to aorto-enteric fistula (AEF) in mediastinal lymphoma is reviewed.     

Iatrogenic secondary post‐partum haemorrhage: Apropos of two uncommon cases

In this article we would like to highlight uterine pseudoaneurysm as a cause of secondary post‐partum haemorrhage following Caesarean section. We would like to stress Doppler ultrasound scan as the initial screening modality for this condition. We also describe angioembolization as the prudent treatment option for this condition rather than resorting to surgery.     

Clinical pharmacology research in the pediatric patient: the challenge continues

For most of the 20th century, most drugs labeled by the United States Food and Drug Administration (USFDA) have not been adequately studied in the pediatric population. This lack of data has necessitated the continued dependence of practitioners on sub-optimal prescribing data placing pediatric patients at great risk of serious therapeutic misadventures. Recently, the USFDA has enacted and begun to enforce the Final Rule of 1997 which became effective on 1 April 1999. This rule is the culmination of the persistent efforts of numerous professional organizations, clinicians, academicians, the USFDA and others, to ensure the ready availability of appropriate data for medications intended for or that will be used in children. Unlike the 1994 Rule which voluntarily required pharmaceutical manufacturers to submit pediatric data, the Final Rule mandates submission of such data and, most importantly, empowers the USFDA to afford incentives and penalties for non-compliance including possible removal of already marketed products. This overview addresses many of the important components which must be included in the performance of a comprehensive clinical pharmacologic evaluation serving as the foundation for optimal dosing across the broad age range encompassing pediatric practice. Furthermore, the possible risk and/or benefits of the study must be reasonably defined prior to undertaking the study and clearly shared with the patient's caregivers. Consent should always be obtained from the caregiver and, when appropriate, assent obtained from the underage child. To facilitate such clinical investigations and to foster collaborative efforts with innovators and clinical research programs, the National Institutes of Health through the National Institute of Child Health and Human Development of the NIH established a network of Pediatric Pharmacology Research Units. These units have worked closely together and with other pediatric research centers to facilitate USFDA labeling of a number of commonly used medications. All of these very positive efforts highlight the many challenges that remain for the pediatric investigator and practitioner while underscoring the very positive environment in support of these efforts.     

Transepithelial paracellular leakiness induced by chronic phorbol ester exposure correlates with polyp-like foci and redistribution of protein kinase C-alpha

Although exposure of LLC-PK1 epithelial cell sheets to phorbol esters (TPA) causes a near immediate and total decrease of transepithelial electrical resistance (TER), continuation of exposure for 3 to 4 days results in a tachyphylactic response as TER begins to return to control levels. Recovery of TER is maximal by 5 to 6 days, but reaches only 70 to 80% of control level. A reciprocal change in the transepithelial flux of D-mannitol indicates that the TER decrease is indicative of an increase in tight junction permeability. Exposure of cell sheets to TPA for several days also results in the appearance of multilayered polyp- like foci (PLFs) across the otherwise one cell layer thick cell sheets. The pattern of penetration of the electron dense dye, ruthenium red, from the apical surface, across the tight junction and into the lateral intercellular space indicates that the tight junctions of the cell sheet become uniformly leaky after acute exposure to TPA. However, when exposure is continued for several days, only the junctions of cells in the PLFs manifest leakiness. The decrease in TER following acute TPA exposure correlates with the translocation of protein kinase C-alpha (PKC alpha) into a membrane-associated compartment. With exposure of several days, only a trace of PKC alpha is visible by Western immunoblot, and this is in the membrane-associated compartment. Immunofluorescent microscopy indicates that the trace of PKC alpha seen in the Western immunoblots is ascribable distinctly to cells of the PLFs. Monolayer areas between PLFs show no discernible immunofluorescent signal. The data therefore indicate that tight junction barrier function may be restored in certain areas by the down regulation of PKC alpha from the membrane-associated compartment. Failure to down regulate may result in the paracellular leakiness and abnormal cell architecture of the PLFs. Possible implications of this model for in vivo epithelial tumor promotion are discussed.      

Sputum tumour necrosis factor-alpha and leukotriene concentrations in cystic fibrosis

It is postulated that a vigorous host inflammatory response in the cystic fibrosis lung contributes to lung injury. Tumour necrosis factor-alpha (TNF-alpha) may play a part in that process and in the generation of leukotrienes. Therefore, the relationships between sputum TNF-alpha, leukotriene concentration, and lung function abnormalities in 16 children with cystic fibrosis were investigated. Each subject provided sputum samples and performed spirometry. TNF-alpha was measured by enzyme linked immunosorbent assay; individual leukotrienes were separated using high performance liquid chromatography and quantified by radioimmunoassay. The geometric mean concentration of TNF-alpha was 129.7 pg/ml and 95% confidence interval 48.2 to 348.3. Mean (SEM) leukotriene B4 (LTB4) was 97.8 (22.9) pmol/g and total cysteinyl leukotrienes were 60.9 (14.8) pmol/g. Mean (SD) forced expiratory volume in one second (FEV1) of the group was 53 (15)% of predicted and forced vital capacity (FVC) was 65 (14)% of predicted. There was a significant positive correlation between TNF-alpha and both LTB4 and the total cysteinyl leukotriene sputum content. An inverse relationship existed between TNF-alpha and FEV1 and FVC. Moreover, a negative correlation was observed between sputum LTB4 and FEV1 and FVC. These results suggest that TNF-alpha and the leukotrienes may participate in the airways inflammation and airflow obstruction observed in cystic fibrosis subjects and support the hypothesis that TNF-alpha upregulates the 5-lipoxygenase pathway in vivo.     

A new amino acid mixture permits new approaches to the treatment of phenylketonuria

A new amino acid mixture for incorporation into medical foods for the treatment of hyperphenylalaninemia has been tested in a regular clinic. The mix is designed to be as unobtrusive as possible, consistent with good nutrition. After more than 1 year of trial as a beverage, we have shown that it is safe and well tolerated but that plasma phenylalanine is no better controlled than with some other products. The mix can be incorporated into a large number of different foods without affecting the taste. Occult monitoring of the quantity of medical foods purchased compared with the amounts reported to be consumed in diet histories provides an excellent way to monitor dietary compliance.