hTFIIIB-beta stably binds to pol II promoters and recruits RNA polymerase III in a hTFIIIC1 dependent way.

It has been shown that under specific conditions, transcription of protein coding genes can be efficiently initiated by RNA polymerase (pol) III in vitro. We examined the formation and composition of such pol III transcription complexes on the duck histone H5 and alphaA-globin promoters and found that the essential step for the formation of pol III transcription complexes on these pol II promoters was the stable binding of transcription factor (TF) IIIB-beta. For this process, the intact TFIIIB-beta complex, consisting of TBP and associated factors (TAFs) was needed and the prior association of pol III assembly factors was not necessary. We demonstrate for the first time that hTFIIIB-beta alone is able to bind to pol II promoter DNA. This resulted in a very stable complex which was resistant to high concentrations of heparin. Although immunodepletion revealed that TBP is essentially required for complex formation, other components of hTFIIIB-beta must also be involved, since TBP itself is unable to form heparin-resistant complexes and does not mediate pol III commitment per se. pol III is recruited to these pol II promoters in a strictly TFIIIC1 dependent way. After binding of TFIIIB-beta, the addition of TFIIIC1 and pol III were sufficient to yield productive pol III transcription complexes, which utilized the correct pol II initiation site. From these findings, we postulate that TFIIIC1 is involved in the recruitment of pol III and may thus form a bridge between TFIIIB-beta and the enzyme. This finding provides the first evidence for functional contacts between TFIIIC1 and pol III, which could be of general importance for the assembly of pol III transcription complexes.

The activity binding to the termination region of several pol III genes represents a separate entity and is distinct from a novel component enhancing U6 snRNA transcription.

Human TFIIIC1, a basal transcription factor essentially required for expression of all pol III genes, exerts its function without primarily binding to DNA. We report here the purification of a termination site binding activity (TBA) which was initially described to be contained in fractions designated as TFIIIC0. TBA specifically and strongly binds to the termination region of pol III genes with internal promoters and can be completely separated from TFIIIC1and a TFIIIC1related activity (TFIIIC1-like), proving that DNA-binding of TBA is independent of these latter activites. Although TBA is not essentially required for, it strongly stimulates pol III transcription from intragenic promoters. This stimulation strictly depends on the presence of TFIIIC1and is not observed in conjunction with TFIIIC1-like. We further present the identification of a novel activity, TFIIIU, which is also contained in crude fractions of TFIIIC0. TFIIIU can be separated from TBA by further purification and is essentially involved in transcription of the mammalian U6 gene. TFIIIU cannot be substituted for by any of the established U6 transcription factors and thus represents a novel U6 transcription factor.

Comparison of the solutions of Bretschneider, St. Thomas' Hospital and the National Institutes of Health for cardioplegic protection during moderate hypothermic arrest.

We evaluated three cardioplegic solutions, Bretschneider's cardioplegic solution (HTK), St. Thomas' Hospital solution (STH) and the solution of the National Institutes of Health (NIH), a solution with added nitroglycerin and lidocaine, for their ability to minimize ischemia-reperfusion injury in a working rat heart model. After cardioplegic arrest at 4 degrees C and subsequent 45 min of ischemic storage at 25 degrees C the function recovery of hearts was examined during 1 h of normothermic crystalloid reperfusion using Krebs-Henseleit buffer as perfusion medium. We noted a significantly better preservation of the maximum (+dp/dt(max)) and minimum (-dp/dt(max)) velocity of pressure development and a significantly higher coronary flow with the use of HTK (2,657 mm Hg/s, 2,122 mm Hg/s, 17 ml/min) compared to STH (1,600 mm Hg/s, p < 0.05; 1,591 mm Hg/s, p<0.05; 11 ml/ min, p<0.05), and an intermediate level of preservation of hemodynamic parameters with NIH (2,149 mm Hg/s, 1,766 mm Hg/s, 12 ml/min). Concerning the cardiac output, however, no major difference was found between the HTK (41 ml/min), the STH (34 ml/min) and the NIH group (36 ml/min). The decay of the myocardial energy charge was significantly lower in both the HTK and the NIH group as compared with conservation in STH solution. Lactate was lowest in the HTK group, CK and LDH releases in the effusate remained lowest after HTK and NIH preservation. The data of this study suggest that HTK and NIH most perfectly reduce the impairment of myocardial function and provide better myocardial protection during ischemic arrest at 25 degrees C and superior recovery compared to STH solution.

A nucleosome positioned in the distal promoter region activates transcription of the human U6 gene.

To investigate the consequences of chromatin reconstitution for transcription of the human U6 gene, we assembled nucleosomes on both plasmids and linear DNA fragments containing the U6 gene. Initial experiments with DNA fragments revealed that U6 sequences located between the distal sequence element (DSE) and the proximal sequence element (PSE) lead to the positioning of a nucleosome partially encompassing these promoter elements. Furthermore, indirect end-labelling analyses of the reconstituted U6 wild-type plasmids showed strong micrococcal nuclease cuts near the DSE and PSE, indicating that a nucleosome is located between these elements. To investigate the influence that nucleosomes exert on U6 transcription, we used two different experimental approaches for chromatin reconstitution, both of which resulted in the observation that transcription of the U6 wild-type gene was enhanced after chromatin assembly. To ensure that the facilitated transcription of the nucleosomal templates is in fact due to a positioned nucleosome, we constructed mutants of the U6 gene in which the sequences between the DSE and PSE were progressively deleted. In contrast to what was observed with the wild-type genes, transcription of these deletion mutants was significantly inhibited when they were packaged into nucleosomes.

Human transcription factors IIIC2 , IIIC1 and a novel component IIIC0 fulfil different aspects of DNA binding to various pol III genes.

Human transcription factor IIIC2 interacts with the TFIIIA-5S DNA complex and forms a ternary TFIIIA/IIIC2-5S DNA complex. Formation of this complex does not preclude simultaneous binding of TFIIIC2to the B-box sequence of a second template. This suggests that the domain(s) or subunit(s) required for indirect recognition of the 5S promoter by TFIIIC2 are different from those necessary for direct binding of TFIIIC2 to B-box-containing pol III promoters. Whereas TFIIIC2 is only required for transcription of the 'classical' pol III genes, TFIIIC1 is generally required for transcription of all pol III genes, including that of the U6 gene. The activity of TFIIIC1 strongly enhances specific binding of basal pol III factors TFIIIA, TFIIIC2 and the PSE binding protein (PBP) to their cognate promoter elements and it acts independently of the corresponding termination regions. Moreover, we characterize an activity, TFIIIC0, purified from phosphocellulose fraction C, which shows strong DNase I protection of the termination region of several pol III genes and which is functionally and chromatographically distinct from TFIIIC1 and TFIIIC2.

HTK versus UW solution for myocardial protection during moderate hypothermia.

The objective of this study was to assess the protective capacity of UW solution in comparison to Bretschneider's (HTK) cardioplegic solution under moderate hypothermic conditions (25 degrees C), as those usually present during intraoperative myocardial protection. Ischemia-induced alterations of cardiac function parameters were analyzed and compared for each solution after 45 min of ischemic storage and 60 min of reperfusion with oxygenated Krebs-Henseleit buffer (KHB), using a rat working-heart model. Compared to nonischemic values, left-ventricular systolic and diastolic pressure, +dp/dtmax and -dp/dtmax were significantly better maintained in the HTK (95 mm Hg, 7 mm Hg, 2,657 mm Hg/s and 2,122 mm Hg/s) than in the UW group (76 mm Hg, p < 0.05, 11 mm Hg, p < 0.05, 1,745 mm Hg/s, p < 0.05 and 1,600 mm Hg/s, p < 0.05). Concerning the myocardial contents of ATP, creatine phosphate and the energy charge, a minor decrease was observed after preservation in HTK compared to UW solution. The results of this study indicate superior myocardial protection with the use of HTK solution for protection of the heart at 25 degrees C compared to UW solution.

Human TFIIIA alone is sufficient to prevent nucleosomal repression of a homologous 5S gene.

Plasmid DNA harbouring the human 5S rRNA gene was assembled into nucleosomes using either Xenopus S150 extracts or purified core histones in the presence of pectin. In both cases reconstitution of nucleosomes led to a complete repression of transcription. This repression could be efficiently counteracted by preincubating the template DNA with highly purified hTFIIIA which allowed the protein to bind to the ICR of the 5S gene. By using an efficient and well-defined in vitro reconstitution system based on isolated core histones in the presence of pectin, which is devoid of endogenous transcription factors, we demonstrate here for the first time that human TFIIIA alone is sufficient to prevent nucleosomal repression of h5S gene transcription and that additional pol III transcription factors are not required to achieve this effect. Additionally, we investigated the binding of hTFIIIA to a mononucleosome reconstituted on the human 5S gene. DNAse I footprinting experiments reveal that the entire ICR of the human 5S gene is covered by the nucleosome, thereby precluding the subsequent binding of human TFIIIA to the promoter of the 5S gene.

Transcription factor IIA is inactivated during terminal differentiation of avian erythroid cells.

Avian histone H5 and alpha A-globin genes are transcribed much more efficiently in whole cell extracts derived from immature polychromatic erythrocytes than in extracts from mature duck erythrocytes. We found that these differential activities are detectable only if assayed with promoters containing a functional TATA box. The addition of either highly purified human or recombinant yeast transcription factor IIA (TFIIA) to extracts from mature erythrocytes resulted in a significant increase in transcription from TATA-containing promoters, whereas transcription from TATA-less promoters remained unaffected. Moreover, the activity of TFIIA was found to be reduced in extracts from mature erythrocytes. These data support the proposition that inactivation of TFIIA may contribute to a general repression of gene activity in avian erythrocytes, and only those genes with alternative mechanisms of initiation complex formation continue to be expressed in these cells. In the case of the histone H5 gene, such an alternative mechanism could be mediated via the interaction between duck erythrocyte upstream stimulating factor and TFIID.

Transcription factor eUSF is an essential component of isolated transcription complexes on the duck histone H5 gene and it mediates the interaction of TFIID with a TATA-deficient promoter.

We analysed the formation of transcription complexes on the H5 gene of the duck which is efficiently transcribed in HeLa cell extracts in vitro. Upon deletion of its TATA-box, the fidelity of transcription of the H5 gene is maintained, although the efficiency of this process is significantly reduced. Selective inactivation of TFIID in whole cell extracts and reconstitution experiments either with human recombinant TFIID or a protein fraction from duck erythrocytes enriched in TFIID show that transcription of the TATA-less H5 promoter nevertheless requires the protein TFIID. Screening of promoter elements which could indirectly mediate the interaction of TFIID with a TATA-less H5 promoter led to the identification of a sequence element located about 40 base-pairs downstream from the H5 initiation site that shows partial homology to the USF consensus sequence. In electrophoretic mobility shift and footprinting studies we demonstrated a specific interaction of the erythroid factor USF (eUSF) with this downstream element. By isolating active transcription complexes we found that all components required for correct initiation remain stably associated with the H5 promoter irrespective of the presence or absence of the TATA box. Moreover, the reconstitution of eUSF and TFIID-depleted transcription complexes with purified protein fractions demonstrate that not only TFIID but also eUSF essentially participates in complex formation even on H5 promoter mutations lacking the TATA-box. Mutual interactions between eUSF and TFIID appear to stabilize the binding of TFIID in the presence or absence of its proper binding site.

Incidence and severity of acute cardiac allograft rejection with two different low-dose cyclosporine maintenance protocols.

Currently cyclosporine (CyA) represents the main immunosuppressive agent used after cardiac transplantation and usually is administered in combination with prednisone and/or azathioprine for prevention of graft rejection. From March, 1984, to August, 1987, 53 patients underwent orthotopic heart transplantation for terminal-stage heart disease at the Second Department of Surgery, University of Vienna. All patients received CyA in increasing dosage (3 mg/kg to 6-10 mg/kg) postoperatively according to renal function, obtaining a trough high-pressure liquid chromatographic whole-blood target level of 200 to 400 ng/ml at the end of the first week. CyA was subsequently tapered to 100 to 150 ng/ml after 6 months. From March, 1984, through April, 1986, maintenance immunosuppression was carried out with a double-drug regimen of CyA and azathioprine. Since May, 1986, a triple-drug schedule was applied with CyA, azathioprine, and prednisone. Under triple-drug therapy, the incidence of mild, moderate (p less than 0.0001), and severe (p = 0.05) allograft rejection proven by endomyocardial biopsy decreased significantly with a corresponding increase of absent (p less than 0.0001) rejection. Freedom from moderate, severe, and lethal graft rejection, number of rejection episodes per patient after 1 year (double drug, 1.0, versus triple drug, 2.5), and patient survival disclosed significant improvement for recipients of the triple-drug regimen. Both groups had the same incidence of infectious complications; freedom from death by infection after 1 year was 90% versus 91% (double versus triple drug, p = 0.20).(ABSTRACT TRUNCATED AT 250 WORDS)

[The radiologic picture of pulmonary sequestration. Evaluation of 11 of our own cases and 123 cases published in the literature]. / Das radiologische Erscheinungsbild der Lungensequester. Auswertung 11 eigener und 123 in der Literatur publizierter Fälle.

Pulmonary sequestration is an extremely rare malformation and most reported series are therefore small. For this reason an attempt has been made to review our own cases and those reported in the literature in order to describe the radiological appearances and to distinguish between the intralobar and extralobar types. 85% of pulmonary sequestrations are localised in the lower zones, with a distinct preference for the left side. 28% of intralobar sequestrations have a homogeneous appearance, 33% are inhomogeneous and 39% are cystic; 77% of extralobar sequestrations are homogeneous and 23% are cystic. Extralobar sequestrations never showed an inhomogeneous appearance. There was a difference in the manifestations of the two forms: 75% of intralobar and 59% of extralobar sequestrations were symptomatic. Distinction between the two types of sequestration could not be based on their arterial supply nor on their venous drainage.