AtPng1 knockout mutant of Arabidopsis thaliana shows a juvenile phenotype, morpho-functional changes, altered stress response and cell wall modifications.

In order to ascertain the role of plant transglutaminases (TGase) in growth and abiotic stress response, the AtPng1 knock out (KO) line of A. thaliana has been analyzed during plant development and under heat and wound stress. Comparing wild type (WT) and KO lines a 58-kDa band was immunodetected by anti-AtPng1p antibody in the cell wall and chloroplasts only in the WT line. A residual TGase activity, not showing correlation with development nor stress response, was still present in the KO line. The KO line was less developed, with a juvenile phenotype characterized by fewer, smaller and less differentiated cells. Chloroplast TGase activity was insensitive to mutation. Data on stressed plants showed that (i) KO plants under heat stress were more juvenile compared to WT, (ii) different responses between WT and KO lines after wounding took place. TGase activity was not completely absent in the KO line, presenting high activity in the plastidial fraction. In general, the mutation affected A. thaliana growth and development, causing less differentiated cytological and anatomical features.

A Zea mays 39-kDa thylakoid transglutaminase catalyses the modification by polyamines of light-harvesting complex II in a light-dependent way.

A transglutaminase (TGase; EC 2.3.2.13) activity, which shared many properties with the TGase activity of the Helianthus tuberosus chloroplast, was observed in the Zea mays L. chloroplast and in its fractions. This activity was found to be prevalent in thylakoids; bis-(glutamyl) spermidine and bis-(glutamyl) putrescine were the main polyamine conjugates formed. Light stimulated the endogenous thylakoid activity. Putrescine, spermidine and spermine were conjugated to the isolated light-harvesting complex of photosystem II (LHCII) with different degrees of efficiency, spermine being the polyamine most efficiently conjugated. A TGase with a light-sensitive activity was identified in the photosystem II-enriched fraction. Its partial purification on a sucrose gradient allowed the separation of a 39-kDa band, which was immunorecognised by two anti-TGase antibodies (Ab-3 and rat prostatic gland-TGase). Both a colorimetric and a radiometric assay for TGase activity, the former carried out in the presence of biotinylated cadaverine and the latter in the presence of polyamines labelled with radioactive isotopes and resulting in the isolation of glutamyl-polyamines, further confirmed that the thylakoid enzyme is indeed a calcium-dependent transglutaminase (Thyl-TGase). At variance with guinea pig liver and erythrocyte TGases, which are insensitive to light, the activity of the thylakoid transglutaminase is affected by light. Moreover, this enzyme, when tested with purified LHCII as substrate, catalysed the production of mono- and bis-glutamyl-polyamines in equal amounts, whereas the 'animal' enzymes produced mainly mono-derivatives. Herein, it is discussed whether this light sensitivity is due to the enzyme or the substrate.

Suborganellar localisation and effect of light on Helianthus tuberosus chloroplast transglutaminases and their substrates.

The light stimulation of transglutaminase (TGase EC 2.3.2.13) activity was verified by incubating isolated chloroplasts of Helianthus tuberosus L. continuously or for alternate periods of light or dark (light/dark and dark/light). The first 10 min of incubation always represented the critical period. Light-harvesting complexes of photosystem II (LHCII) were more intensely labelled by (14)C-polyamines under light and light/dark than under dark and dark/light conditions. Chloroplasts were fractionated into thylakoid- and stroma-enriched fractions in which multiple TGase forms and substrates were found. Antibodies against TGase recognised 58- and 24-kDa bands in thylakoids and a 150-kDa band in the stroma. The latter, and its 150-kDa fraction, catalysed the conjugation of 14C-polyamines to Rubisco. In both fractions (thylakoid-pre and stroma-pre) the analysis of polyamine glutamyl derivatives showed a significant light-affected conjugation of polyamines to endogenous proteins. Alternatively, entire chloroplasts were incubated and afterwards their sub-fractions were isolated (thylakoid-post and stroma-post). The PSII and LHCII complexes were more intensely immunodetected in thylakoid-post than in thylakoid-pre, especially under dark conditions. Conversely, the conjugation of polyamines to thylakoid proteins was clearly light-stimulated in thylakoid-post, and much less in thylakoid-pre. Stroma-pre proteins were poorly polyamine-conjugated and not light-affected; on the contrary, stroma-post proteins were much more polyamine-modified and strongly light-stimulated. Thus, the light-activated conjugation depends mainly on the presence of the thylakoid fraction during the assay. The protective effect on chloroplasts under photo-damage, stress or senescence conditions attributed in the literature to free polyamines is discussed with regard to the occurrence of polyamine conjugates catalysed by TGases.

Transglutaminase activity during senescence and programmed cell death in the corolla of tobacco (Nicotiana tabacum) flowers.

Corolla life span of undetached flowers of Nicotiana tabacum was divided into stages from the closed corolla (stage 1) through anthesis (stage 5) to death (stage 9). Senescence began around stage 6 in the proximal part, concomitantly with DNA laddering. Nuclear blebbing, DNA laddering, cell wall modification, decline in protein, water, pigment content and membrane integrity were observed during senescence and PCD. Transglutaminase activity was measured as mono- and bis-derivatives of putrescine (mono-PU; bis-PU) and bis-derivatives of spermidine (bis-SD). Bis-derivatives decreased with the progression of senescence, while mono-PU increased during early senescence; derivatives were present in different amounts in the proximal and distal parts of the corolla. In excised flowers, exogenous spermine delayed senescence and PCD, and caused an increase in free and acid-soluble conjugated PA levels. Bis-PU was the most abundant PA-derivative before DNA laddering stage; thereafter, bis-PU generally decreased and mono-PU became the most abundant derivative.

[Hemodynamic and coronary angiographic performance at a center with medium-low activity: organization model, activity indicators, and costs]. / Performance emodinamica e coronarografica in un centro a medio-basso volume di attività: modello organizzativo, indicatori di attività e costi.

BACKGROUND: In order to evaluate the cost-effectiveness of coronary angiography performed in a low volume Center, we examined our 1-year activity. METHODS: The organizational model of the multipurpose cardiac catheterization laboratory is described. In this type of facility both coronary angiographic and electrophysiological studies are performed. To evaluate the laboratory performance we examined the utilization level, the appropriateness of the studies, the complication rates and the number of studies that had to be repeated because of inadequate data or image quality. The costs were calculated for the in-house laboratory setting (the actual scenario) and for the 25 km distant laboratory setting (the historical scenario). RESULTS: The laboratory caseload of coronary angiography was 342 studies, 46% of the overall laboratory activity; 175 patients (51%) underwent non-pharmacological therapy, 129 patients (38%) were treated with medical therapy; the percentage of patients with normal coronary arteries was 11%. Two patients (0.58%) had vascular complications, 1 patient (0.29%) developed an acute myocardial infarction 2 hours after coronary angiography without any evidence of angiographic modifications at the repeated study. In no patient the study had to be repeated because of inadequate data or image quality. The mean cost of a coronary angiography was Lit. 512,000 (265 Euro) for the actual scenario; it would have been Lit. 694,000 (359 Euro) for the historical scenario, with Lit. 182,000 (94 Euro) saved. CONCLUSIONS: These findings are consistent with the accepted criteria of good laboratory performance and cost-effectiveness. Thus coronary angiography can be performed effectively and efficiently in a low volume Center.

[Effects of filtering techniques on time-domain analysis of signal-averaged ECG after acute myocardial infarction: a multicenter study, GISS-3 arrhythmia sub-project]. / Effetti delle techniche di filtraggio sull'analisi nel dominio del tempo del signal-averaged ecg dopo infarto miocardico acuto: studio multicentrico, GISSI-3 sottoprogetto aritmie. GISSI-3 Sottoprogetto Aritmie (Elenco dei Centri in ultima pagina).

AIM OF THE STUDY: To evaluate the influence of different filtering techniques on the measurement of ventricular late potentials (VLP) the Sottoprogetto Aritmie of GISSI-3 collected signal-averaged ECG (SAECG) from 647 patients. METHODS: Data were recorded after myocardial infarction (10 +/- 4 days) in 20 Italian Coronary Units. Three main filtering algorithms were used in the different commercial devices: Bidirectional Filter (ART, Aerotel, Fidelity Medical) (BF: 340 Patients), Spectral Filter (Marquette) (SF: 258 Patients) and Del Mar Filter (Del Mar Avionics) (DF: 49 Patients). QRS duration (QRSD), low amplitude signal duration (LAS40) and root mean-square-voltage (RMS40), were measured with various filters set at 40-250 Hz high and low pass frequencies. RESULTS: After correction for clinical variables the measurements of VLP in the three different groups were different. QRSD value obtained by BF (100.6 +/- 13 ms) was shorter than that obtained by SF (109.1 +/- 12 ms). No differences were found in LAS40 and RMS40 values between SF and BF, while DF gave longer LAS40 and lower RMS40 than SF and BF. Residual noise was lower in BF (0.3 +/- 0.1 muV). than in SF and DF (0.5 +/- 0.1 muV). Applying standard criteria DF gave a higher prevalence of VLP (48.9%) than BF (23.8%) and SF (19%) groups. CONCLUSIONS: This study demonstrates that the use of different filters produces discordant result on VLP measurements. For correct application of SAECG analysis in risk stratification after myocardial infarction, normal and abnormal values must be specifically established for the different filter techniques.

[Comparison of vectorcardiography and echocardiography in the evaluation of the site and extent of necrosis in myocardial infarction without Q wave]. / Confrontation vectocardiographie-échocardiographie dans l'évaluation du siège et de l'étendue de la nécrose dans les infarctus myocardiques sans onde Q.

In order to improve the evaluation of the site and extent of the necrosis in acute myocardial infarction without Q wave (IMNQ), we used a multiparametric method taking into account the enzymatic values of the total CK, a score derived from Frank's VCG and a kinetic score of the VG segments estimated by echo-2D. The literature data have shown, according to the clinical and anatomical correlations, the frequency of the transmural lesions in the IMNQ cases and, on the contrary, of the lesions limited to the endocardial region and partially transmural in infarctions with new pathological Q waves. The heterogeneousness of the anatomo-pathological data can be explained by the mechanisms of the ischaemia, the site of the coronary occlusion, the presence or not of a spasm, the compensation by the collateral flux and eventually the effects of a treatment by fibrinolytic drugs.

[Histological substrate of ventricular tachycardias of the right bundle-branch block type (sensitive to verapamil)]. / Substrato istologico delle tachicardie ventricolari tipo blocco di branca destra (sensibili al verapamil).

In order to clarify the anatomic substrate of idiopathic ventricular tachycardia of right bundle-branch block morphology, responsive to verapamil, two patients with this type of arrhythmia were evaluated using hemodynamic studies, right ventricular endomyocardial biopsy as well as electrophysiological and pharmacological studies. Pressures, right and left ventriculography and coronary angiography were normal in both patients. Case 1 showed a prolonged sinus node recovery time and a histological pattern which was compatible with healed myocarditis. Case 2 presented a complete right bundle-branch block, intra-His conduction defects and histological features of aspecific cardiomyopathy. Our results underline the presence of diffuse ventricular histologic changes in patients with idiopathic ventricular tachycardia of right bundle-branch block morphology. These histologic changes seem unable to interfere with cardiac pump function but are sufficient to create significant abnormalities of impulse formation and conduction.

Right precordial ST and QRS changes in the diagnosis of right ventricular infarction.

Two groups of patients with anatomically proved acute myocardial infarction were compared in order to study specificity and sensitivity of the ECG criteria previously described in clinical and experimental right ventricular infarction ( RVI ). Group 1 included 21 patients with left inferior infarction and with a variable degree of right ventricular involvement; group 2 included nine patients with myocardial infarction confined to the left inferior wall. In both groups the presence of ST elevation (at least 0.05 mV) and the morphology of the QRS complex in V4R , V3R, and V1 were assessed in ECGs performed at the time of admission. Also, in order to evaluate the morphology of the ST segment and QRS complex in right precordial leads in normal subjects, an ECG with 12 standard and four right precordial leads ( V6R to V3R) was performed in 82 subjects (group 3) without clinical and ECG evidence of heart disease. Our data reveal that in normal subjects an rS pattern is always present in V3R and frequently (91%) in V4R . On the contrary, the presence of QS or QR complexes in both V4R and V3R are specific markers of right ventricular necrosis (specificity 100%; sensitivity 78%). The presence of injury and necrosis waves in V4R or V4R to V3R during inferior infarction is a useful diagnostic criterion in that it insures a highly specific diagnosis of acute RVI in the great majority (76 and 71%, respectively) of the cases with autopsy evidence of right ventricular involvement.