Endogenous protein phosphorylation by resting and activated human neutrophils

NADPH oxidase is an enzyme in the plasma membrane of the neutrophil that catalyzes the production of O2-, a species central to the oxygen- dependent killing mechanisms of this cell. The oxidase is dormant in resting cells and is activated upon the addition of a stimulus. Neutrophils of patients with chronic granulomatous disease (CGD) manifest no oxidase activity when stimulated. The possible role of protein phosphorylation in the activation of NADPH oxidase was examined in normal and CGD neutrophils by measuring the incorporation of 32Pi into proteins as determined by gel electrophoresis followed by autoradiography. Resting neutrophils from normal subjects exhibit at least 40 distinct phosphoprotein bands. The level of phosphorylation of these bands was examined after the addition of phorbol myristate acetate (PMA), opsonized zymosan, digitonin, N-formyl-methionyl- phenylalanine (FMLP), or NaF. PMA and opsonized zymosan increased the phosphorylation of a set of 6 protein bands. Digitonin and FMLP consistently caused the phosphorylation of 4 of these protein bands, while NaF failed to induce increased phosphorylation of any protein band. All activators tested caused the dephosphorylation of one specific protein band. The time course of phosphorylation (dephosphorylation) was examined using PMA as the activating agent. Increased phosphorylation of one protein band was evident by 12 sec after the addition of PMA. The most slowly phosphorylated protein band did not slow evidence of change until 5 min after the addition of PMA. Three of the phosphoproteins examined were phosphorylated either earlier than or concomitant with the activation of NADPH oxidase. CGD neutrophils were compared with normal cells for their ability to phosphorylate proteins in response to PMA. The phosphoprotein banding patterns of CGD neutrophils were identical with those of normal neutrophils in both the resting and activated states. The evidence presented shows that the phosphorylation of proteins is a prominent feature of neutrophil metabolism. The striking similarity of phosphorylation changes induced by the various activators tested suggests that protein phosphorylation may play a role in some aspects of neutrophil activation. Evidence was not obtained, however, regarding a link between protein phosphorylation and activation of NADPH oxidase.     

Role of plasminogen activator inhibitor-1 in promoting fibrin deposition in rabbits infused with ancrod or thrombin

The role of defective fibrinolysis caused by elevated activity of plasminogen activator inhibitor-1 (PAI-1) in promoting fibrin deposition in vivo has not been well established. The present study compared the efficacy of thrombin or ancrod, a venom-derived enzyme that clots fibrinogen, to induce fibrin formation in rabbits with elevated PAI-1 levels. One set of male New Zealand rabbits received intravenous endotoxin to increase endogenous PAI-1 activity followed by a 1-hour infusion of ancrod or thrombin; another set of normal rabbits received intravenous human recombinant PAI-1 (rPAI-1) during an infusion of ancrod or thrombin. Thirty minutes after the end of the infusion, renal fibrin deposition was assessed by histopathology. Animals receiving endotoxin, rPAI-1, ancrod, or thrombin alone did not develop renal thrombi. All endotoxin-treated rabbits developed fibrin deposition when infused with ancrod (n = 4) or thrombin (n = 6). Fibrin deposition occurred in 7 of 7 rabbits receiving both rPAI-1 and ancrod and in only 1 of 6 receiving rPAI-1 and thrombin (P < .01). In vitro, thrombin but not ancrod was inactivated by normal rabbit plasma and by purified antithrombin III or thrombomodulin. The data indicate that elevated levels of PAI-1 promote fibrin deposition in rabbits infused with ancrod but not with thrombin. In endotoxin-treated rabbits, fibrin deposition that occurs with thrombin infusion may be caused by decreased inhibition of procoagulant activity and not increased PAI-1 activity.     

Tricuspid annulus motion and mitral annulus motion: Anatomical intimacy causing a good correlation?

BACKGROUND:

Echocardiographic evaluation of the heart and its function, especially left ventricular systolic function, has great clinical importance. Systolic function can be measured using several methods, such as the amplitude of motion of the left atrioventricular plane (mitral annulus motion [MAM]) toward the apex during systole. Similarly, right ventricular systolic function can be measured using the motion of the right atrioventricular plane (tricuspid annulus motion [TAM]) toward the apex during systole.

OBJECTIVES:

Because the mitral and tricuspid annuli are situated close to each other in the fibrous skeleton between both ventricles and atria, one might think that a decrease in the amplitude of MAM would be followed by a decrease in the amplitude of TAM. The present study was developed to determinine if this anatomical intimacy causes a good correlation between the amplitudes of TAM and MAM.

METHODS:

Nineteen healthy subjects and 103 consecutive patients were included in the study and examined using echocardiography. The amplitudes of TAM and MAM were measured and the correlation between the amplitudes was calculated.

RESULTS:

In the 103 consecutive patients, a significant but relatively weak positive correlation was found between TAM and MAM amplitudes (Pearson’s correlation coefficient [r]=0.58; P<0.001). In the 19 healthy subjects, no significant correlation was found.

CONCLUSIONS:

Despite the anatomical intimacy of the annuli, the correlation between the amplitudes of TAM and MAM in consecutive patients was rather weak, and there was no correlation in healthy subjects. These findings could be due to anatomical and physiological differences between the right and left ventricles.     

Evolution of the genus Leishmania as revealed by comparisons of nuclear DNA restriction fragment patterns.

Restriction endonuclease DNA fragment patterns have been used to examine the relationships among 28 isolates of Leishmania as well as Crithidia, Endotrypanum, and Trypanosoma cruzi. Fragments of nuclear DNA were generated with six restriction enzymes, and blots were hybridized with probes from three loci. Among the major lineages the fragment patterns are essentially completely different, while within the major lineages various degrees of divergence are found. Molecular evolutionary trees were constructed using the method of Nei and Li to estimate the percent nucleotide sequence divergence among strains from the fraction of fragments shared. Defined groups, such as species or subspecies within the major lineages, are also grouped by nuclear DNA comparisons. Within the donovani complex, we find Leishmania donovani chagasi and Leishmania donovani infantum to be as similar as strains within Leishmania donovani donovani, consistent with the proposal by other workers that New World visceral leishmaniasis originated quite recently.     

Changes in the sensitivity of adult cockroach corpora allata to a brain allatostatin

There are major changes in the sensitivity of corpora allata from the cockroach Diploptera punctata toward the allatostatic tridecapeptide APSGAQRLYGFGL-amide (ASAL) during the female reproductive cycle, as revealed by measurement of juvenile hormone (JH) biosynthesis in vitro. Glands from recently molted adult females show only 30-40% inhibition at 10 nM ASAL, falling to a minimum of less than 10% on day 5 at the peak of spontaneous JH synthesis in vitro. The decline in JH synthesis observed in post-vitellogenic females is accompanied by a dramatic increase to ca. 90% ASAL sensitivity at 10 nM by day 6. Then sensitivity slowly wanes during subsequent ovulation and pregnancy to the levels typical of previtellogenic and virgin females. Full dose/response studies reveal a second level of response at ca. 1 microM, which resembles the pattern obtained from whole brain extracts. We conclude that physiological sensitivity to ASAL (IC50 ca. 0.1 nM) is correlated with the preparation for choriogenesis, and we suggest that 1000 times higher doses give a cross-reaction with related allatostatic receptor(s) that confer important sensitivity at other development stages.     

Précising definitions as a way to combat overdiagnosis

Roughly, overdiagnosis (ODx) occurs when people are harmed by receiving diagnoses (often accompanied by interventions) that do not benefit them, usually because the diagnosed conditions do not pose a threat to their health. ODx is a theoretical as well as a practical problem as it relates to definitions of disease. Elsewhere, it has been argued that disease is a vague concept and that this vagueness may contribute to ODx. In response, we develop a stipulative or précising definition of disease, for the specific purpose of decreasing or preventing ODx. We call this disease_ODx, aimed at distinguishing cases where it would be beneficial to identify (and treat the condition) from those where diagnosis is more likely to harm than benefit. A preliminary definition of disease_ODx is that X is a disease_ODx iff there is dysfunction that has a significant risk of causing severe harm. This paper examines the 3 concepts in this definition, using a naturalistic account of function, a Feinbergian account of comparative harm, and a probabilistic understanding of risk. We then test the utility of this approach using examples of clinical conditions that are currently overdiagnosed.     

Caged-in: Successful percutaneous closure of left atrial appendage with Watchman-FLX in the presence of proximal thrombus

Percutaneous mechanical closure of the left atrial appendage (LAA) is a valuable stroke prevention strategy in patients with atrial fibrillation and contraindication to oral anticoagulation. LAA thrombus is a common finding in patients with atrial fibrillation and frequently fails to resolve despite therapeutic anticoagulation. In this scenario, LAA occlusion device implant is generally discouraged due to the high risk of thrombus dislodgement and embolization; however, alternative management options are limited. We report the first case of a successful LAA occlusion device (Watchman-FLX) implant in the presence of a proximal thrombus.