An extended conformation of calmodulin induces interactions between the structural domains of adenylyl cyclase from Bacillus anthracis to promote catalysis.

The edema factor exotoxin produced by Bacillus anthracis is an adenylyl cyclase that is activated by calmodulin (CaM) at resting state calcium concentrations in infected cells. A C-terminal 60-kDa fragment corresponding to the catalytic domain of edema factor (EF3) was cloned, overexpressed in Escherichia coli, and purified. The N-terminal 43-kDa domain (EF3-N) of EF3, the sole domain of edema factor homologous to adenylyl cyclases from Bordetella pertussis and Pseudomonas aeruginosa, is highly resistant to protease digestion. The C-terminal 160-amino acid domain (EF3-C) of EF3 is sensitive to proteolysis in the absence of CaM. The addition of CaM protects EF3-C from being digested by proteases. EF3-N and EF3-C were expressed separately, and both fragments were required to reconstitute full CaM-sensitive enzyme activity. Fluorescence resonance energy transfer experiments using a double-labeled CaM molecule were performed and indicated that CaM adopts an extended conformation upon binding to EF3. This contrasts sharply with the compact conformation adopted by CaM upon binding myosin light chain kinase and CaM-dependent protein kinase type II. Mutations in each of the four calcium binding sites of CaM were examined for their effect on EF3 activation. Sites 3 and 4 were found critical for the activation, and neither the N- nor the C-terminal domain of CaM alone was capable of activating EF3. A genetic screen probing loss-of-function mutations of EF3 and site-directed mutations based on the homology of the edema factor family revealed a conserved pair of aspartate residues and an arginine that are important for catalysis. Similar residues are essential for di-metal-mediated catalysis in mammalian adenylyl cyclases and a family of DNA polymerases and nucleotidyltransferases. This suggests that edema factor may utilize a similar catalytic mechanism.

Syk- and Lyn-dependent phosphorylation of Syk on multiple tyrosines following B cell activation includes a site that negatively regulates signaling.

The Syk protein tyrosine kinase is an essential component of the B cell Ag receptor signaling pathway. Syk is phosphorylated on tyrosine following B cell activation. However, the sites that are modified and the kinases responsible for these modifications have yet to be determined. To approach this problem, we used a mapping strategy based on the electrophoretic separation of peptides on alkaline polyacrylamide gels to identify the tryptic phosphopeptides derived from metabolically labeled Syk. In this work, we report that Syk from activated B cells is phosphorylated principally on six tyrosines: one located between the tandem SH2 domains (Tyr130); three in the linker region (Tyr317, Tyr342, and Tyr346); and two in the catalytic domain (Tyr519 and Tyr520). The linker region sites are the primary targets of the Src family protein tyrosine kinase, Lyn, and include a site that negatively (Tyr317) regulates receptor signaling. Efficient phosphorylation of the catalytic domain and inter-SH2 domain tyrosines is catalyzed primarily by Syk itself, but only occurs to an appreciable extent in cells that express Lyn. We propose that these sites are phosphorylated following the binding of Syk to immunoreceptor tyrosine-based activation motif.

[Pulmonary hypertension in acute pulmonary embolism]. / Plucna hipertenzija u akutnoj emboliji pluca.

Twenty-five patients with acute pulmonary embolism without other pulmonary or heart diseases were analyzed for pulmonary hypertension. Doppler echocardiography was used to determine the systolic pressure of the pulmonary artery (PAPs) from the maximal velocity of the tricuspid regurgitation using corrected Bernoulli's formula (PAPs = 1.23 x 4 Vmax2 - 0.09). Pulmonary hypertension was found in 84% (21/25) of the patients with acute pulmonary embolism. PAPs values ranged between 34 and 90 mmHg (X = 54 +/- 7.5 mmHg) and hypocapnia with carbon dioxide partial pressure, PaCO2, ranged from 26 to 34 mmHg (X = 30 +/- 2 mmHg). PAPs showed a significant negative correlation with oxygen partial pressure (r = -0.87, P < 0.01). According to the findings of lung scintigraphy, all patients with pulmonary hypertension had submassive pulmonary embolism with perfusion abnormalities in two segments (X = 5 +/- 2 segments). It is concluded that pulmonary hypertension may be expected in more than 80% of the patients with submassive acute pulmonary embolism, and hypoxemia and hypocapnia. Doppler echocardiography is a noninvasive method useful in the diagnosis and follow-up of pulmonary hypertension in patients with acute pulmonary embolism.

[Doppler echocardiography in the evaluation of pulmonary hypertension in chronic obstructive pulmonary disease]. / Doplerska ehokardiografija u procjeni plucne hipertenzije kod kronicne opstrukcijske plucne bolesti.

Systolic pulmonary arterial pressure (PAPs) at rest and during submaximal exercise using bicycle ergometer was estimated in 80 patients with clinical stable obstructive pulmonary disease (COPD). Systolic pulmonary arterial pressure was estimated by using continuous Doppler from maximal velocity of the tricuspid regurgitant jet by applying the modified Bernoulli equation and regression: PAPs = 1.23 x 4 Vmax2-0.09 (mmHg). Pulmonary hypertension (PAPs (m) 30 mmHg at rest, or PAPs (o) 40 mmHg during the exercise) was registered in 56% of the patients with chronic obstructive pulmonary disease. Thirty percent of the patients (24/80) had mild degree latent pulmonary hypertension (PAPs (o) = 41-47 mmHg), and 26% (21/80) of the patients with COPD had mild to moderate manifest pulmonary hypertension (PAPs(m) = 33-47 mmHg). In 11 patients with manifest pulmonary hypertension (52%), a decrease of PAPs for 5 mmHg or more was registered after oxygen-test. These patients were grouped as responders. Systolic pulmonary arterial pressure at rest and during the exercise has best correlation with arterial PaO2 (r = -0.73 to -0.87), and out of parameters of pulmonary ventilation it has the best correlation with index FEF50/FVC (r = -0.49 to -0.68). By using continuous wave Doppler systolic pulmonary artery pressure was estimated in 84% of the patients with COPD (80/95). Fifteen patients were excluded from the study because of the bad echocardiographic window or inadequate Doppler tricuspid regurgitation signal.

[Evaluation of the reliability of endogenous creatinine clearance as in indicator of renal function in ambulatory patients. I]. / Ocena pouzdanosti klirensa endogenog kreatinina kao pokazatelja bubrezne funkcije u ambulantnih bolesnika (I. Deo).

Clearance of the endogenous creatinine is a simple method for application, it is relatively cheap and applicable in almost all surroundings, but it is not reliable enough when the obtained results are concerned. This report shows the results of determining this clearance in comparison with radiosotope clearance 51 cr-EDTA, referent for clinical research, in 52 outpatients with primary and secondary kidney diseases. It was found that values of clearance of the endogene creatinine (Clkr) are significantly lower than the clearance 51 Cr-EDTA (ClEDTA) (t = 2.391, p < 0.01) and that the correlation between the two method is poor (r = 0.688). The source of mistakes should be looked for (and strictly controlled) when gathering diuresis, because the biochemical method of creatnine concentration determination of the biologic material itself is reliable enough.

[Echocardiography in the diagnosis of cardiac sarcoidosis]. / Ehokardiografija u dijagnostici sarkoidoze srca.

This paper deals with echocardiographic changes in the heart in patients with active intrathoracic sarcoidosis. Heart changes were registered with 10 (38%) out of 26 patients, in all radiological stages of sarcoidosis. Out of 16 patients first radiological stage (BHL) was found with 4 (25%), out of 8 patients second radiological stage was found with 4 (25%), and third radiological stage was found with 2 patients. Out of 10 patients with echocardiographic changes in the heart 4 patients (40%) had increased echoes and hypokinesis of the proximal part of interventricle septum, 3 (30%) had significantly reduced diffusive capacity and pulmonary hypertension. Increased echoes of papillary muscles were registered in two cases, one of them with relatively mitral insufficiency. In one case dilatational incipient cardiomyopathy with second stage of relatively mitral insufficiency was found and in one case increased echoes and hypokinesis of the back wall of the left ventricle were registered. Changes in electrocardiogram were found with 5 (19%) patients suffering from pulmonary sarcoidosis.

[The effect of acute bronchospasm on pulmonary artery pressure]. / Utjecaj akutnog bronhospazma na vrijednosti tlaka u pulmonalnoj arteriji.

The influence of acute airways obstruction on the values of the systolic pulmonary arterial pressure in patients with bronchial asthma is presented. The acute airways obstruction has been attained by the nonspecific bronchial provocation test on methacholine, using the modified Cockcroft and associates method. The systolic pulmonary arterial pressure has been determined using the non-invasive Doppler echocardiograpfhy method from the corrected Bernoulli and associates equation (GP = 4V2). The mild acute airways obstruction has been provoked in 11 cases, with the 28% fall of average values of the forced one-second expiratory volume (FEV1) and the 48% flow-volume curve of the forced expiratory flow at 50% of the vital capacity (FEF50). A moderate hypoxemia has also been registered with the 22% fall of the average values of the arterial oxygen tension (PaO2). At the same time, a considerable increase of the systolic pulmonary arterial pressure (PAPs) was recorded, with the average values of 31 to 39 mmHg (mean = 33.7 mmHg) which corresponds to a mild pulmonary arterial hypertension. The PaO2 at PAPs values show a considerable negative correlation (r = s -0.73; p < 0.01). The Doppler echocardiography is the most sensitive noninvasive method suitable for diagnosis and observing the pulmonary arterial hypertension in patients with bronchial asthma.

Thoracocentesis under ultrasonographic control.

Chest ultrasonography has been advocated as an effective tool for diagnosis and localization of pleural effusion. To determine if chest ultrasonography would be beneficial, when thoracocentesis was performed, 71 patients with suspected pleural effusion were evaluated. Two groups of patients were formed according to the presence of small or large suspected effusion ascertained by chest posteroanterior roentgenogram. Chest ultrasound was found very useful, both for establishing diagnosis of pleural fluid and for obtaining adequate fluid samples of small effusions. This technique had no such advantage during thoracocentesis of large effusions. During and after thoracocentesis, there were no complications in any group of patients. Authors concluded that ultrasound method seems to be very useful in choosing the optimal site for thoracocentesis, particularly of small effusions.

[Lymphangiomyomatosis of the lungs]. / Limfangiomiomatoza pluca.

Pulmonary lymphangiomyomatosis (LAM) is a rare disease at the women's child bearing age, characterized by the proliferation of smooth muscles along the lymphatics in the lung, in mediastinal and retroperitoneal lymph nodes as well as in retroperitoneal lymph vessels. The disease manifests itself as a progressive dyspnea, chylous pleural effusions and reccurent pneumothorax. Uncured the disease progresses to a serious respiratory insufficiency and death within a period of ten years since the manifestation of the first symptoms. We studied a female patient in whom the diagnosis of pulmonary lymphangiomyomatosis was established on clinical findings and pathohistologic analysis of biopsy material. The patient was offered progesterone which, according to some authors, stops further progression of the disease. The progesterone treatment was carried out for a year and it is going to be continued. During the period mentioned, repeatedly controlled lung diffusing capacity and arterial blood gas analysis remained basically unchanged compared to these before treatment. It is noteworthy, that once formed changes in the parenchyma are irreversible, so early diagnosis and timely started treatment are of basic importance.

[Ultrasound in the diagnosis of pulmonary and pleural diseases]. / Ultrazvuk u dijagnostici bolesti pluca i pleure.

Ultrasound as a method in evaluation of pleural diseases, peripheral pulmonary infiltrates and tumors has already been accepted throughout the world. However we haven't yet been applying it widely. In this work the possibilities of ultrasound as a method in pleural and lung diseases diagnostics have been described, together with the typical findings and examination technique. Indications of thoracic ultrasound which along with unavoidable X-ray examinations represent a new increase of quality in the diagnostics of pleural and lung diseases are specified.