Mitotic repression of transcription in vitro.

A normal consequence of mitosis in eukaryotes is the repression of transcription. Using Xenopus egg extracts shifted to a mitotic state by the addition of purified cyclin, we have for the first time been able to reproduce a mitotic repression of transcription in vitro. Active RNA polymerase III transcription is observed in interphase extracts, but strongly repressed in extracts converted to mitosis. With the topoisomerase II inhibitor VM-26, we demonstrate that this mitotic repression of RNA polymerase III transcription does not require normal chromatin condensation. Similarly; in vitro mitotic repression of transcription does not require the presence of nucleosome structure or involve a general repressive chromatin-binding protein, as inhibition of chromatin formation with saturating amounts of non-specific DNA has no effect on repression. Instead, the mitotic repression of transcription appears to be due to phosphorylation of a component of the transcription machinery by a mitotic protein kinase, either cdc2 kinase and/or a kinase activated by it. Mitotic repression of RNA polymerase III transcription is observed both in complete mitotic cytosol and when a kinase-enriched mitotic fraction is added to a highly simplified 5S RNA transcription reaction. We present evidence that, upon depletion of cdc2 kinase, a secondary protein kinase activity remains and can mediate this in vitro mitotic repression of transcription.

Nuclear assembly with lambda DNA in fractionated Xenopus egg extracts: an unexpected role for glycogen in formation of a higher order chromatin intermediate.

Crude extracts of Xenopus eggs are capable of nuclear assembly around chromatin templates or even around protein-free, naked DNA templates. Here the requirements for nuclear assembly around a naked DNA template were investigated. Extracts were separated by ultracentrifugation into cytosol, membrane, and gelatinous pellet fractions. It was found that, in addition to the cytosolic and membrane fractions, a component of the gelatinous pellet fraction was required for the assembly of functional nuclei around a naked DNA template. In the absence of this component, membrane-bound but functionally inert spheres of lambda DNA were formed. Purification of the active pellet factor unexpectedly demonstrated the component to be glycogen. The assembly of functionally active nuclei, as assayed by DNA replication and nuclear transport, required that glycogen be pre-incubated with the lambda DNA and cytosol during the period of chromatin and higher order intermediate formation, before the addition of membranes. Hydrolysis of glycogen with alpha-amylase in the extract blocked nuclear formation. Upon analysis, chromatin formed in the presence of cytosol and glycogen alone appeared highly condensed, reminiscent of the nuclear assembly intermediate described by Newport in crude extracts (Newport, J. 1987. Cell. 48:205-217). In contrast, chromatin formed from phage lambda DNA in cytosol lacking glycogen formed "fluffy chromatin-like" structures. Using sucrose gradient centrifugation, the highly condensed intermediates formed in the presence of glycogen could be isolated and were now able to serve as nuclear assembly templates in extracts lacking glycogen, arguing that the requirement for glycogen is temporally restricted to the time of intermediate formation and function. Glycogen does not act simply by inducing condensation of the chromatin, since similarly isolated mitotically condensed chromatin intermediates do not form functional nuclei. However, both mitotic and fluffy interphase chromatin intermediates formed in the absence of glycogen can be rescued to form functional nuclei when added to a second extract which contains glycogen. This study presents a novel role for a carbohydrate in nuclear assembly, a role which involves the formation of a particular chromatin intermediate. Potential models for the role of glycogen are discussed.

Mitotic repression of RNA polymerase III transcription in vitro mediated by phosphorylation of a TFIIIB component.

Interphase cytosol extracts prepared from Xenopus laevis eggs are active in RNA polymerase III (Pol III) transcription. Addition of recombinant B1 cyclin to these extracts activates mitotic protein kinases that repress transcription. Affinity-purified p34cdc2-cyclin B kinase (mitosis-promoting factor) is sufficient to effect this repression in a simplified Pol III transcription system. This mitotic repression involves the direct phosphorylation of a component of the Pol III transcription initiation factor TFIIIB, which consists of the TATA box-binding protein (TBP) and associated Pol III-specific factors. The transcriptional activity of the TFIIIB-TBP fraction can be modulated in vitro by phosphorylation with mitotic kinases and by dephosphorylation with immobilized alkaline phosphatase.

Cardiac rupture in embolic myocardial infarction after implantation of a valved aortic conduit.

We report a case of an apical myocardial infarction complicated by left ventricular wall rupture due to coronary artery embolism four years after surgery of an acute type A dissection of an aortic aneurysm with implantation of a valved aortic conduit.

[Soft-tissue reconstruction of the dorsum of the hand and finger to cover the extender tendons]. / Weichteilrekonstruktion am hand- und fingerrücken über dem streckapparat.

Small defects of extensor tendons can mostly be covered by local flaps. For larger defects the groin flaps, pedicled flaps from the forearm and free flaps are indicated. Between January 2001 and December 2005, we performed the following pedicled and free flaps to reconstruct the soft tissue defects of the hand or distal forearm: 23 pedicled flaps: 15 posterior interosseus artery or perforator flaps, 8 radial forearm flaps; 26 free flaps: 4 radial forearm flaps, 12 lateral upper arm flaps, 10 latissimus, serratus fascia or scapula flaps. With this study we want to illustrate the different procedures, compare their surgical techniques and outcomes and finally to rate the results. In the context of security, morbidity of the donor site and functional outcomes, our favourite flap is the lateral upper arm flap.

Modulation of L-selectin ligand expression during an immune response accompanying tumorigenesis in transgenic mice.

Immune surveillance depends on lymphocyte access to tissue. Lymphocytes emigrate from blood when adhesion receptors such as L-selectin and the alpha 4 beta 7 integrin on these cells bind to ligands expressed on venular endothelium. Among transgenic mouse lines expressing an oncoprotein (Tag) in islet beta cells, some recognize Tag as nonself. In these mice, Tag expression elicits both beta cell hyperplasia with subsequent progression to tumors and lymphocytic infiltration. Endothelial ligands for L-selectin and alpha 4 beta 7 were upregulated in infiltrated islets in these transgenic mice. These ligands were not expressed in tumors, which were devoid of lymphocytic infiltration. In contrast, the adhesion molecules PECAM-1, ICAM-1, and VCAM-1 were expressed on endothelium in both noninfiltrated tumors and infiltrated islets. Thus, upregulation of expression of endothelial ligands for L-selectin and alpha 4 beta 7 may contribute to autoimmune infiltration. Repression of expression of these same ligands may be involved in the failure of tumor immunity.

Generation of a variant t(2;8) translocation of Burkitt's lymphoma by site-specific recombination via the kappa light-chain joining signals.

We analyzed the Burkitt's lymphoma line BL64 in which a reciprocal translocation joins the immunoglobulin kappa light-chain locus on chromosome 2 to the c-myc gene on chromosome 8. The breakpoints on the two marker chromosomes 8+ and 2p- occurred 5' of the Js segment within the conserved nonamer and heptamer recombination sequences. Both signals were detected directly adjacent to the breakpoints in sequences of chromosome 8 suggesting that the translocation in BL64 was catalyzed by enzymes normally involved in V-J recombination. The distance between the c-myc gene and the breakpoint in J kappa amounts to at least 90 kilobases on the DNA level. In one allele of the c-myc gene somatic mutations were found in the promoter-leader region. This allele is transcribed and is supposed to be involved in the translocation. The half-life of the c-myc-specific mRNA in BL64 cells is not prolonged in comparison to the normal c-myc message. These results suggest that in Burkitt's lymphoma the translocation occurs during an early stage of B-cell differentiation and that in the variant translocations mechanisms other than a prolonged half-life, such as changes in transcriptional rates, or other posttranscriptional RNA processing contribute to the high steady-state level of c-myc RNA in the cytoplasm.

Role of maturation-promoting factor (p34cdc2-cyclin B) in differential expression of the Xenopus oocyte and somatic-type 5S RNA genes.

Transcription of 5S rRNA and tRNA genes by RNA polymerase III (pol III) in cytosolic extracts of unfertilized Xenopus eggs and in a reconstituted system derived from Xenopus oocytes is repressed by the action of one or more mitotic protein kinases. Repression is due to the phosphorylation of a component of the pol III transcription apparatus. We find that the maturation/mitosis-promoting factor kinase (MPF, p34cdc2-cyclin B) can directly mediate this repression in vitro. Affinity-purified MPF and immune complexes formed with antibodies to the protein subunits of MPF (p34cdc2 and cyclin B) retain both histone H1 kinase activity and the capacity to repress transcription in the reconstituted transcription system. Transcription complexes of oocyte-type 5S RNA genes and tRNA genes are quantitatively more sensitive to MPF repression than the corresponding transcription complexes of the somatic-type 5S RNA gene. The differential transcription of oocyte- and somatic-type genes observed during early Xenopus embryogenesis has been reproduced with the reconstituted transcription system and affinity-purified MPF. This differential transcription may be due to the instability of transcription complexes on the oocyte-type genes and the heightened sensitivity of soluble transcription factors to inactivation by mitotic phosphorylation. Our results suggest that MPF may play a role in vivo in the establishment of the embryonic pattern of pol III gene expression.

Three breakpoints of variant t(2;8) translocations in Burkitt's lymphoma cells fall within a region 140 kilobases distal from c-myc.

The variant translocations t(2;8) in Burkitt's lymphoma cells join band q24 of chromosome 8, distal from c-myc, to the Igkappa locus, with considerable variation in the location of the breakpoints on chromosome 8. We report the cloning and molecular characterization of a chromosome 8 region, distal from the c-myc locus, which encompasses the breakpoints of the Burkitt's lymphoma cell lines BL64, BL21, and LY91 within 11 kilobase pairs, termed provisionally bvr-1 (Burkitt's variants' rearranging region 1). Using probes from the c-myc, the bvr-1, and the human pvt-1 loci obtained by chromosome walking coupled with pulsed-field gel electrophoresis, we have constructed a physical map of the region 3' of c-myc. We map bvr-1 and pvt-1 about 140 and 260 kilobase pairs, respectively, distal from c-myc.

Spin-labelled analogues of GDP and GTP as site-specific reporter groups for guanosine nucleotide-binding proteins.

New derivatives of GDP and GTP have been synthesized for the spectroscopic investigation of the interaction between guanosine nucleotides and guanosine nucleotide-binding proteins. The 3'-hydroxyl group in these nucleotides was replaced by a 3'-amino group, which was further derivatized by the introduction of a spin-label reporter group. The biological activity of 3'SL-GDP and 3'SL-GTP could be demonstrated by measuring the interaction of these spin-labelled derivatives with bacterial elongation factor Tu. The amino modification and spin labelling only slightly influenced the affinity of the guanosine nucleotides for EF-Tu from Escherichia coli or Thermus thermophilus. Electron paramagnetic resonance (EPR) measurements revealed a strong immobilization of the labelled nucleotides upon binding to T. thermophilus EF-Tu. Significant differences between the spectra of EF-Tu X 3'SL-GDP, EF-Tu X 3'SL-GTP and aminoacyl-tRNA X EF-Tu X 3'SL-GTP ternary complexes were observed. Our data demonstrate that spin-labelled guanosine nucleotides can be used as sensitive spectroscopic probes for the investigation of the local environment of the nucleotide-binding site during distinct functional states of a guanosine nucleotide-binding protein.

Expression of synthetic cDNA sequences encoding human insulin-like growth factor-1 (IGF-1) in the mammary gland of transgenic rabbits.

We have developed an expression system where foreign proteins are synthesized specifically in the mammary gland of transgenic rabbits and secreted into the milk. Regulatory elements were isolated from the bovine alpha S1-casein-encoding gene and combined with a synthetic DNA coding for human IGF-1 and for [Gln58]IGF-1, an IGF-1 analogue. The resulting hybrid DNA constructs were used to generate transgenic rabbits. Females of seven transgenic lines tested were positive for synthesis of IGF-1. Transmission of the transgene to progeny and IGF-1 production in female offspring was observed in all transgenic lines analysed. As expected, expression of transgene mRNA could only be detected in the mammary gland. Production levels of transgenic protein were as high as 1 g IGF-1 per liter rabbit milk. IGF-1, as well as [Gln58]IGF-1, when secreted into rabbit milk, was correctly processed and biologically active. IGF-1 was purified from the milk of transgenic rabbits to a nearly homogenous active form.

Cardiac tamponade following acupuncture.

We present a rare complication of acupuncture in a 83-year-old woman who developed syncope and cardiogenic shock shortly after an acupuncture procedure into the sternum. Echocardiography revealed cardiac tamponade, and pericardiocentesis disclosed hemopericardium. Due to hemodynamic instability, thoracotomy was indicated. A small but actively bleeding perforation of the right ventricle was found and successfully closed. Although acupuncture represents a relatively safe therapeutic intervention, this case report should remind all acupuncturists of possible and sometimes life-threatening adverse effects.

VATS (video-assisted thoracic surgery) of undefined pulmonary nodules. Preoperative evaluation of videoendoscopic resectability.

Peripheral undefined pulmonary nodules have become a favorable indication for the videoendoscopic approach in thoracic surgery. In our latest experience, we also successfully applied this technique in centrally located lesions of the lung. In reviewing our first 29 cases, we looked for preoperative features of videoendoscopic resectability. From March 1992 to September 1993, 29 patients underwent videothoracoscopy for undefined pulmonary nodules at our hospital. This group consisted of 17 men and 12 women (aged 25 to 77 years). Pulmonary nodules of this group of patients were defined as centrally located when close attachment to the segmental or subsegmental bronchopulmonary unit was observed and/or the distance to the visceral pleura exceeded 10 mm. Nodules that did not meet any of these criteria were hence interpreted as peripheral lesions. In the course of 21 excisions of peripheral lesions, we had to convert to open thoracotomy only once for anatomic reasons. When using the video-assisted thoracic surgery (VATS) approach for centrally located lesions, we succeeded in removing four of six. We failed only if the lesions were located in the upper lobe but could easily apply the technique for centrally located lesions in the lower lobes. In conclusion, undefined peripheral pulmonary nodules are a favorite indication for VATS. Centrally located pulmonary nodules of the lower lobes can often be managed easily by VATS, especially if the interlobar fissure extends to the stem of the pulmonary artery. Centrally located pulmonary nodules in the upper lobes may not be suitable for the VATS approach due to the special anatomic arrangement of the upper lobe segmental arteries and bronchioles.

Videoendoscopic removal of a mediastinal cyst.

A 41-year-old woman was admitted to the hospital for obstetric surgery. A preoperative chest x-ray film showed a mediastinal mass. After examinations with echocardiography, computed tomography, and magnetic resonance imaging, we removed a cyst that was 2.7 x 3.5 cm in size by thoracoscopic means. The patient left the hospital 3 days after the operation.

Valvuloplasty with glutaraldehyde-treated autologous pericardium in patients with complex mitral valve pathology.

BACKGROUND: Severe mitral regurgitation associated with complex mitral valve disease often precludes successful surgical repair. The feasibility and the results of valvuloplasty with glutaraldehyde-treated autologous pericardium remain largely unknown. METHODS: The cases of 63 patients who underwent operation within an 11-year period were studied. A pretreated autologous pericardial patch was used for leaflet extension plasty, for paracommissural plasty, as a substitute for part of the leaflet, and for reimplantation of ruptured papillary muscles to eliminate severe mitral regurgitation. Patients with a severely calcified annulus after en bloc decalcification had straddling endoventricular pericardial patch annuloplasty for reconstruction of the affected atrioventricular groove. Chordal replacement with a strip of pericardium was chosen if no suitable chordae were available. Pericardium-reinforced suture annuloplasty was used in patients with acute endocarditis resistant to medical therapy. Associated valvuloplasty procedures with Carpentier techniques were also employed. RESULTS: There were no operative deaths in this series. At a mean follow-up of 61.1 months (range, 4 to 132 months), mitral regurgitation was absent or trivial in 92.1% of patients by echocardiography. Freedom from reoperation was 95.2% at 1 year and 5 years. Thromboembolic events have not been detected. Thirty percent of patients returned to sinus rhythm. Two patients required valve replacement. CONCLUSIONS: Our beneficial results indicate that glutaraldehyde-treated autologous pericardium is suitable for valvuloplasty. It provides durable and predictable repair of valves that might otherwise need to be replaced because of the complex mitral valve disease. The technique is reliable, allows further efficacious repair possibilities, and improves postoperative outcomes. Whether it can prevent late deterioration and calcification requires more investigation.

Valve repair in mitral regurgitation complicated by severe annulus calcification.

BACKGROUND: Valvuloplasty has significant advantages over valve replacement for mitral regurgitation, but the presence of severe calcification of the mitral valve apparatus has been thought to preclude successful valve reconstruction in general. The purpose of this report is to assess the results of valvuloplasty in patients with severe mitral regurgitation having extensive calcification extending from the mitral annulus to underlying myocardium and parts of the papillary muscles. METHODS: Thirty-seven adult patients with severe mitral regurgitation and calcification were operated on between April 1990 and January 1998. Twenty-six patients had degenerative disease, 4 had acute bacterial endocarditis, 6 had postrheumatic fever, and 1 patient had Marfan's disease. The valve repair comprised of en bloc decalcification with extensive leaflet debridement and reconstruction of the annulus. Autologous pericardium was used in patch-extended endocardial annuloplasty or leaflet repair. Valve competence was retained after correction of regurgitation by sliding atrioplasty, rotation paracommissural sliding plasty, cusp remodeling, or chordal repair. All patients required a prosthetic annuloplasty. RESULTS: Follow-up echocardiography at 47 months (range, 3 to 92 months) showed no or only trivial mitral regurgitation in 33 patients; 3 had grade I-II mitral regurgitation and 1 required valve replacement after 3 months. Freedom of reoperation at 1 and 5 years was 94.6%. At last examination, 33 patients were in New York Heart Association functional class I and 3 in class I-II; there has been no mortality and no thromboembolic events. CONCLUSIONS: Valvuloplasty can be safely and successfully carried out in patients suffering from regurgitation associated with severe calcification of the mitral apparatus. With encouraging beneficial midterm results, we suggest patients with calcified valves should not be excluded from mitral repair.

Prophylaxis of thromboembolism in general surgery: comparison between standard heparin and Fragmin.

In a double blind randomized trial on 250 patients undergoing elective abdominal surgery the effect of Fragmin was compared with heparin. Patients over 40 years of age except appendectomy and herniotomy were included. The dose of heparin was 5,000 IU b.i.d. whilst Fragmin was given in a dose of 2,500 U once per day and the second injection was a placebo. Prophylaxis started 2 h preoperatively and was maintained for 7 days. The fibrinogen uptake test was used as a screening method for thrombosis which was confirmed by phlebography. 124 patients were in the heparin group and 126 in the Fragmin group. Comparability between groups was found in: age, sex, Broca index, amount and type of risk factors, type of surgery. Thromboembolism was found in 10 cases in each group. Blood transfusions on the postoperative days 1 to 6 were required in 2 Fragmin and in 12 heparin patients. The total amount of blood given during that time was 6 units in the Fragmin and 37 units in the heparin group. These differences were significant.

Early investigation of silver-coated Silzone heart valves prosthesis in 126 patients.

BACKGROUND AND AIM OF THE STUDY: Permanent silver (Silzone) coating of the sewing cuff of St. Jude Medical prosthetic heart valves may reduce the rate of prosthetic valve endocarditis (PVE). However, the incidence of paravalvular leaks and stroke in patients after implantation of Silzone-coated heart valve prostheses is largely unknown. METHODS: Complications were analyzed among 126 consecutive patients (78 males, 48 females; mean age 64.7 years; range: 40-80 years) who received Silzone-coated prostheses at our institution between February 1998 and December 1999. Among patients, 94 had aortic valve replacement, 29 mitral valve replacement, and three had aortic and mitral valve replacement. Concomitant cardiac procedures (mainly coronary bypass) were performed in 47 patients (37.3%). RESULTS: Hospital mortality was 1.6% (2/126) and freedom from valve-related mortality 99.2+/-0.9%. Total follow up was 137.4 patient-years, and 98.4% complete. Strokes or transitory ischemic attacks (TIAs) occurred in four cases (two strokes, two TIAs; stroke rate 1.5%/year; 95% CI 0.4-2.6%/year; stroke+TIA rate 3%/year; 95% CI 0.4-5.25%/year). There was no case of prosthetic valve dysfunction or PVE. Freedom from reoperation due to procedure-associated complications was 97.6% (one aortic dissection, two major bleeds). Paravalvular leak was detected in 19 cases, and graded trivial or mild in 17 cases (11 grade 0-I, six grade I), and moderate in two cases (grade I-II in one, grade II in one). No patient needed reoperation due to paravalvular leak. Left ventricular (LV) function was normal in 80 cases, but was impaired mildly in 20 cases, moderately in 16, and severely in eight. Bicycle exercise testing in 92 patients (73%) showed median exercise performance (81.4+/-23.9% of normal) after correction for age and weight. Cerebral magnetic resonance imaging was performed in 64 patients (50.8%); median MRI score was 2.0+/-1.8 according to a 12-scaled score system. CONCLUSION: The overall incidence of echocardiographic paravalvular leak graded more than trivial or mild was low (1.6%) in patients followed for a mean of 1.1+/-0.5 years (range: 1-27 months) after valve replacement with Silzone-coated heart valve prostheses. No patient required reoperation due to paravalvular leak; moreover, the stroke rate (1.5%/year) was low in these patients. The present data indicated no significant disadvantages or higher rate of complications related to silver coating compared with other, non-silver-coated prostheses. Future investigation is needed to determine long-term outcome.

Early results of cryopreserved pulmonary allografts as aortic valve substitute.

Excellent clinical results with pulmonary autografts and experimental evidence that the pulmonary valve can withstand the higher stress in the systemic circulation led us to use the cryopreserved pulmonary allograft for aortic valve replacement. From September 1988 to March 1991, 45 consecutive patients (59.9 +/- 12.0 years, 25 men and 20 women) received a cryopreserved pulmonary allograft in the aortic position from our hospital based valve bank. All allografts were inserted freehand in the subcoronary position. There were 3 in-hospital deaths (7%) and 1 patient had severe valvular incompetence immediately postoperatively requiring reoperation after 4 weeks. Forty-one patients were followed at 3-6 month interval for 14.7 +/- 7.8 months (3-28 months) and valve performance was assessed routinely by means of color flow Doppler echocardiography: 34 patients (83%) had no or trivial aortic valve regurgitation. Valvular incompetence class II was present in 2 patients (5%) whereas 3 (7%) demonstrated class II-III. Severe aortic regurgitation (class III-IV) could be detected in 2 patients (5%). Both had to undergo reoperation 4 months and 15 months, respectively, postoperatively. Macroscopic and histological evaluation of the explanted valves demonstrated absence of significant degeneration. We assume that a mismatch in size between allograft and aortic annulus could have lead to dilatation of the allograft valve ring and consequently to valvular incompetence. Pulmonary cryopreserved allografts achieve acceptable short-term results which can be improved if initial technical problems can be avoided.

Evaluation of profundoplasty in reconstructions of combined aorto-iliac and femoro-popliteal occlusive disease.

The results in 135 patients undergoing aorto-iliac and aorto-femoral bypass during 1974-1979 were examined. In most cases, occlusive disease of the iliac arteries was combined with total atherosclerotic occlusion of the superficial femoral arteries and sometimes with additional pathology in the profunda femoris artery. In 18.7%, subsequent vascular procedures became necessary in extremities of patients who had undergone aorto-iliac or aorto-femoral anastomotic procedures without additional profundoplasties. In those cases where the primary reconstruction was combined with profundoplasty the results were very satisfying and subsequent procedures were needed only in 8.8%. The aorta-profunda femoris bypass is now preferred for patients with combined aorto-iliac and femoro-popliteal occlusive disease. Results and technical details are discussed.