A novel stopped-flow method for measuring the affinity of actin for myosin head fragments using μg quantities of protein

Summary The dissociation constant for actin binding to myosin and its subfragments (S1 & HMM) is 1 m at physiological ionic strength. Many of the methods used to measure such affinities are unreliable for a K_d below 0.1 m. We show here that the use of phalloidin to stablise F-actin and fluorescently labelled proteins allows the affinity of actin for myosin S1 to be measured in a simple transient kinetic assay. The method can be used for K_d's as low as 10 nm and we demonstrate that the K_d's can be estimated using only g quantities of material. Furthermore we suggest how this method may be adapted for ng quantities of protein. This will allow the affinity of actin for myosin fragments to be estimated for proteins which are difficult to obtain in large quantities i.e. from biopsy material or from proteins expressed in baculovirus.     

Co-Stimulatory Effects of Misoprostol and Diclofenac on Glycosaminoglycan Synthesis in Human Cartilage Explants and Their Therapeutic Relevance

Misoprostol, a prostaglandin E(1) analog, is currently available to manage ulcer disease, being used predominantly in the prophylaxis of nonsteroidal anti-inflammatory drug (NSAID)-induced ulceration, a serious side effect of anti-inflammatory therapy in arthritis. The protective effects of misoprostol have now also been shown to extend to cartilage in a series of experiment using an ex vivo system employing normal human and osteoarthritis (OA) cartilage. Misoprostol reproducibly reverses inhibition of proteoglycan synthesis induced by interleukin-1 and certain NSAIDs, and also stimulates synthesis in OA cartilage. The article reviews these findings and also presents the results obtained in a study of 20 OA cartilages in which synergy was demonstrated between misoprostol (at 50 ng/ml) and diclofenac (0.3 &mgr;g/ml) in preventing proteoglycan synthesis. Diclofenac on its own is unusual amongst NSAIDs in exerting virtually no deleterious effect on cartilage. The synergy with misoprostol is of clinical interest in view of the recent introduction of a misoprostol/diclofenac combination product (Arthrotec), the intention of which was to provide antiinflammatory efficacy with a reduced incidence of GI damage. The implications of these cartilage experiments is that such a combination may also offer improvements in the management of the arthritic process in OA, and methods whereby this would be assessed clinically are discussed.     

The use of the iSTAT portable analyzer in patients undergoing cardiopulmonary bypass

Objective. To evaluate the utility of the iSTAT blood analyzer, a bedside device for hematocrit, sodium, potassium, and glucose measurement during cardiopulmonary bypass (CPB).Methods. Forty patients scheduled for elective CPB were evaluated prospectively. In addition to using the iSTAT analyzer, blood samples were analyzed at four time points: following induction of anesthetic, 10 min. after initiation of CPB, 60 min. after initiation of CPB, and following heparin neutralization by protamine. Blood glucose concentration was measured by the hospital laboratory using a Kodak Analyzer and by a glucose meter, electrolytes were evaluated by the Kodak Analyzer and BGE (a device which is commonly used for satellite laboratory determinations of electrolyte and blood gas results), and hematocrit samples were measured by the hospital laboratory using an NE 8,000 and a centrifuge. The means and standard deviations of the differences between the methods were calculated.Results. The hematocrit values determined by the iSTAT machine, when adjusted for the level of total protein (according to manufacturer's directions), differed from the laboratory values by 0.53 = 1.46 percentage points. An alternative to measuring total protein and making the adjustment is simply adding 1 % to the hematocrit in the pre-CPB period and 3% on-CPB or post-CPB, which we found to yield values that differed from the laboratory by 0.52 ± 1.42 percentage points. For all four tests (hematocrit, sodium, potassium, and glucose) the iSTAT had a similar relationship to the laboratory values as did the other commonly used means (centrifuge, BGE, and glucose meter) of clinical evaluation.Conclusion. In summary, we found that in patients undergoing CPB, the iSTAT values agreed sufficiently well with standard laboratory values and that the iSTAT instrument can be relied upon for bedside measurements.Presented in part at the Annual Meeting of the Society of Cardiovascular Anesthesiologists, Philadelphia, PA, May 1995, and at the 70th meeting of the IARS, Washington DC, March 1996. Supported in part by a grant by iSTAT Corporation.     

Multivariate Analysis of Left Ventricular Mass Determinants in Adults: Different Patterns in Men and Women

Echocardiographic left ventricular mass (LVM) estimates are strong predictors of subsequent mortality and cardiovascular events. It is known that blood pressure (BP), weight (WT), and age are significantly correlated with LVM. We hypothesized that stroke volume (SV) measured by Doppler echocardiography would also be correlated with LVM. Two hundred and thirteen patients referred for routine echocardiography had determination of LVM, cuff BP, and Doppler SV. Those with localized LV disease, valvular disease, or cor pulmonale were excluded. In both men and women, systolic BP (SBP) was more closely correlated with LVM than was diastolic blood pressure or mean arterial pressure, and SV was more closely correlated with LVM than cardiac output or cardiac index. Stepwise regression, followed by multiple regression showed that four variables (WT, SV, SBP, and AGE) explained 32.3% of the variability in LVM in men and 48.5% of the variability in LVM in women. WT and SV were significant determinants of LVM in both men and women. Age was also significant in men and SBP was also significant in women. For both men and women, SV was more significantly correlated with LVM than was SBP. The changes in LVM associated with 1 SD increments of SV and SBP, respectively, were 8 and 5 g for men and 13 and 11 g for women. We conclude that men and women have different patterns of variables influencing LVM. Doppler echocardiographic SV is a newly described determinant of LVM that has a greater correlation with LVM than does SBP. This study reemphasizes the importance of WT as the major determinant of LVM. (ECHOCARDIOGRAPHY, Volume 13, January 1996)     

Monoamine oxidase-dependent metabolism of dopamine in the striatum and substantia nigra of

This article will review the current treatment of pediatric patients with diffuse pontine gliomas (DPG) and discuss three potential avenues of therapeutic research including (i) radiotherapy (RT) in combination with radiation sensitizers, (ii) dose-intensive, induction chemotherapy with hematopoietic support followed in sequence with RT applied as a consolidation therapy, and (iii) the interleafed application of phase-specific chemotherapeutic agents and hyperfractionated external beam radiotherapy (HFEBRT) referred to as chemoradiotherapy.     

Strategies in the treatment of diffuse pontine gliomas: the therapeutic role of hyperfractionated radiotherapy and chemotherapy

This article will review the current treatment of pediatric patients with diffuse pontine gliomas (DPG) and discuss three potential avenues of therapeutic research including (i) radiotherapy (RT) in combination with radiation sensitizers, (ii) dose-intensive, induction chemotherapy with hematopoietic support followed in sequence with RT applied as a consolidation therapy, and (iii) the interleafed application of phase-specific chemotherapeutic agents and hyperfractionated external beam radiotherapy (HFEBRT) referred to as chemoradiotherapy.     

Antimetastatic action of the prostacyclin analogue cicaprost in experimental mammary tumors

Summary In breast cancer, the survival rate strongly depends on the number of lymph nodes involved. A drug with a specific inhibitory activity on lymph node and organ metastases would therefore be a candidate for adjuvant therapy after surgery. Prostacyclin and its stable analogues have been shown to interfere with certain steps of the metastatic cascade and to inhibit the number of lung colonies after i.v.-inoculation of various tumor cell lines. Our data reveal that cicaprost, a metabolically stable and orally active analogue of prostacyclin, has pronounced antimetastatic effects in a series of spontaneously metastasizing rodent tumors. In the SMT 2a and 13762 MTLn3 mammary carcinomas of the rat, cicaprost given daily from the day of tumor implantation strongly inhibits the number of lung metastases as well as lymph node weights without exerting an effect on the primary tumor. Even starting treatment when palpable primary tumors are present gives a pronounced antimetastatic activity. To demonstrate that cicaprost has an effect on metastases already settled in the respective organ, treatment was started after surgical removal of the primary tumor. In the SMT 2a tumor, a strong inhibition of the number of metastases was shown. Interestingly, a perioperative treatment schedule was also effective in both models used. As primary tumor growth in vivo or proliferation in vitro remained unchanged by cicaprost, its mode of action seems to be related to one or more mechanisms of the metastatic process. In tumor cell lines expressing a functional prostacyclin receptor, stimulated tumor cell migration is inhibited and changes of differentiation status are obvious. In conclusion, cicaprost strongly inhibits lymph node and organ metastases of spontaneously metastasizing rodent mammary tumors with a mode of action different from cytostatic or antihormonal drugs.Presented at the symposium "New Approaches in the Therapy of Breast Cancer", Georgetown University Medical Center, Washington DC, October 1994, generously supported by an education grant from Bristol-Myers Squibb.     

Prostate cancer old problems and new approaches

Rates of prostate cancer (PCa) have increased so dramatically over the last decade that the age adjusted incidence rate for PCa is now greater than that any other cancer among men in the United States. This review, published as a three part series, provides a state-of-art assessment of the PCa problem in its divergent aspects.Part 1 covers epidemiology, incidence and progression. Several epidemiological studies have demostrated that first degree male relatives of men with PCa are at increased risk of developing the disease. Familial and genetic factors as well as medical, anthropometric, dietary, hormonal and occupational factors involved in PCa are discussed. Postmortem examination of the prostate in men without evidence of PCa documented a high frequency of adenocarcinoma. Latent disease occurred as early as the second decade of life. Although there is no significant difference in incidence between Caucasian and African-American males, high grade prostatic intraepithelial neoplasia (HGPIN) is higher in the latter group. While dietary fat, androgens and certain environmental factors may be determinants for PCa, the exact mechanism of tumorigenesis is still relatively unknown. The current thinking of the role of genomic instability, chromosomal alterations, tumor suppressor genes and the androgen receptor are explored.