[Left-sided Chylothorax and Tumor of the Right Anterior Mediastinum]. / Linksseitiger Chylothorax und Tumor des rechten vorderen Mediastinums.

An 82-year-old male patient presented with a left-sided spontaneous chylothorax. A computed tomographic scan of the chest revealed a cystic tumor in the right upper anterior mediastinum and nodules in the left mediastinum that we interpreted as enlarged lymph nodes. On suspicion of lymphoma and for therapy of chylothorax, we performed a left-sided thoracotomy. The putative lymph nodes proved to be chylomas. We interpret the right-sided mediastinal tumor as cystic lymphangioma causing the left-sided chylothorax at the junction of thoracic duct and venous system on the right side.

[Diabetes increases short- and long-term mortality after transcatheter aortic valve implantation (TAVI)]. / Diabetes mellitus erhöht die eingriffsbedingte und die langfristige Mortalität nach kathetergestützter Aortenklappenimplantation.

BACKGROUND AND AIM: Long-term mortality after transcatheter aortic valve implantation (TAVI) in elderly patients with abundant comorbidities is considerable. We aimed to determine the impact of diabetes on short- and long-term mortality after TAVI. METHODS: Our study includes 300 consecutive patients (mean age, 82 ± 5 years) who underwent TAVI (158 transapical, 142 transfemoral procedures). All patients were followed by regular telephone contacts. 36% suffered from diabetes. RESULTS: Diabetes could be identified as significant predictor of short- and long-term mortality after TAVI. In diabetic patients, 30-day-mortality was 2,5 fold elevated (18.3% vs. 7.3%, p = 0.004). Furthermore, they were at significantly higher risk of peri-interventional stroke (p = 0.04), stage 3 acute kidney injury (p = 0.003), and prolonged ventilation (p = 0.01). Even after successful TAVI and discharge from hospital, long-term mortality was significantly elevated in diabetic patients (56% vs. 30%, p < 0.0001). Of note, 25% of diabetic vs. only 8% of non-diabetic patients died from cardiac causes during follow-up, suggesting that TAVI is not able to reduce cardiac-related mortality risk in diabetic patients to the same extent as in non-diabetics. CONCLUSION: Diabetes represents a powerful predictor of adverse early and late outcome after TAVI. These findings should be incorporated into the assessment of the risk-to-benefit ratio of TAVI in diabetic patients.

Transposition of greater omentum in deep sternal wound infection caused by methicillin-resistant Staphylococci, with differing clinical course for MRSA and MRSE.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus epidermidis (MRSE) are an increasing problem in deep sternal wound infections (DSWI) after cardiac surgery. METHODS: Between 2005 and 2009, recalcitrant methicillin-resistant Staphylococcus was found in 21 patients with complicated DSWI, and a transposition of the greater omentum (TGO) was finally performed. A positive microbial culture at the time of procedure was present in all patients. The hospital course was reviewed discretely for MRSA and MRSE. RESULTS: Median patient age was 72.3 years (range 60.8-79.7); 76 % of patients were male. Time from the first sternal revision until consecutive open wound therapy due to re-infection and total hospital stay was longer for MRSA compared to MRSE (38 vs. 14 days, P = 0.003, and 141 vs. 91 days, P = 0.007, respectively). The period from cardiac surgery to TGO was likewise prolonged for MRSA (78 vs. 55 days, P = 0.045), whereas in-hospital mortality and one-year mortality rate did not differ. CONCLUSION: TGO remains a good treatment option for DSWI type IV. Microbial findings determine the clinical course; nevertheless in-hospital mortality remains low for both MRSA and MRSE infection.

Minimally invasive partial inferior sternotomy for congenital heart defects in children.

AIM: Minimally invasive approaches for repair of congenital heart defects have gained in popularity. Aim of the study was to evaluate the safety and efficiency of the partial inferior sternotomy approach to repair various congenital heart defects. METHODS: Since 1998, 100 children (55 males; mean age: 3.8 ± 3.7; mean weight: 15.1 ± 8.7 kg) were operated on via a limited median vertical skin incision and partial inferior sternotomy. Preoperative diagnoses were: ASD II (N.=46), sinus venosus defect with partial anomalous pulmonary venous connection (N.=12), partial AV-canal (N.=4), VSD (N.=35), tetralogy of Fallot (N.=2), and double chambered right ventricle (N.=1). Cannulation was always performed via the chest incision. RESULTS: There were no deaths. Mean cross-clamp time was 49.9 ± 30.6 minutes, and mean operation time 192 ± 46 minutes. Mean postoperative mechanical ventilation time, Intensive Care Unit stay and hospital stay were 9.7 ± 10.4 hours, 1.8 ± 0.7 days, and 12 ± 3.0 days, respectively. Complications included pneumothorax requiring drainage in 2 patients, atrioventricular block necessitating a permanent pacemaker in 1 patient. The incisions healed properly. All patients are in excellent condition after a mean follow-up of 32 ± 25 months. On echocardiography no residual defect was evident in 98 patients, and a mild mitral insufficiency in two patients operated on partial atrioventricular canal. CONCLUSION: The partial inferior sternotomy approach to congenital heart operations is less invasive than and cosmetically superior to full sternotomy with reduced postoperative pain and discomfort for the patients. This approach ensures a safe procedure with excellent exposure without additional incisions. It is our standard approach in infants/children with septal defects.

Histological changes in a model of chronic heart failure induced by multiple sequential coronary microembolization in sheep.

AIM: Valuable models of chronic heart failure to perform histological studies are scarce. The authors aimed at investigating histological changes of the heart, lung, liver and kidneys in a stable and reproducible animal model of chronic heart failure in sheep. METHODS: In 8 sheep (N.=8, 77+/-2 kg) chronic heart failure was induced by multiple sequential microembolization through bolus injection of polysterol microspheres (90 microm, N=25 000) into the left main coronary artery. Microembolization (ME) was repeated up to three times in two to three week intervals until animals started to develop stable signs of heart failure. Therefore, clinical and hemodynamic parameters were measured (Troponin T, heart and respiratory rate, cardiac output) after each embolization. Clinical examination was carried out by a veterinarian. All animals were followed for 3 months after first microembolization and then euthanized for histological examination. Histological data of the heart, lung, liver and the kidneys were analyzed in hematoxylin-eosin (HE) stains (10x, 25x, 100x) at baseline (control group) and at 3 months after first ME. Additionally preparations of heart tissue were stained with Picro-Sirius-Red (PSR) for planimetric quantification. A score from 0 to 4 according to Rassler et al. (2005) was used to assess the degree of lung injury. RESULTS: All animals developed histological signs of heart failure as indicated by island-like, patchy fibrosis of the heart. Planimetric quantification (PSR stain) of the heart revealed a significant increase of the total amount of fibrosis from 8+/-2% (base) to 21+/-4% (3 months) (P<0.05), which was distributed homogeneously throughout the left ventricle (20+/-3% left ventricular [LV] anterior wall, 21+/-4% LV posterior wall, 20+/-4% septum). Histologic analysis of the lung demonstrated a moderate degree of interstitial edema and pronounced peribronchial processes of inflammation with beginning proliferation of fibrotic tissue. Liver tissue showed histological changes in terms of pericentral adiposis as sign of hypoxia in course of lacking perfusion. Signs of liver congestion could be detected histological in form of central-venous accumulation of erythrocytes and dissolution of liver tissue in proximity of the central veins. Kidney preparations illustrated loss of endothelial function and vascular occlusions, caused by microspheres, with decline of renal parenchyma particularly of the tubules. CONCLUSION: Multiple sequential intracoronary microembolization can effectively induce myocardial dysfunction with histological signs of chronic ischemic cardiomyopathy and pathological changes of lung, liver and kidney, which can directly be coursed by chronic heart failure. Thus, the present model may be suitable in experimental work on heart failure and LV assist devices, e.g. for studying the impact of mechanical unloading, mechanisms of recovery and reverse remodeling.

Chronic heart failure induced by multiple sequential coronary microembolization in sheep.

OBJECTIVE: Although a large variety of animal models for acute ischemia and acute heart failure exist, valuable models for studies on the effect of ventricular assist devices in chronic heart failure are scarce. We aimed to establish a stable and reproducible animal model of chronic heart failure in sheep. METHODS: Sheep (n=8, 77 +/- 4 kg) were anesthesized and a 5F sheath was implanted into the left carotid artery. The left main coronary artery was catheterized under flouroscopic guidance and bolus injection of polysterol microspheres (90 microm, n=25.000) was performed. Microembolization (ME) was repeated up to three times in two to three week intervals until animals started to develop stable clinical signs of heart failure. Clinical and echocardiographic data were analyzed at baseline (base) and at three months (3 mo) after first ME. All animals were followed for 3 months after first microembolization and then sacrificed for histological examination. Another four healthy sheep (79+/-6 kg) served as control animals. RESULTS: All animals developed clinical signs of heart failure as indicated by increased heart rate at rest (68+/-4 bpm (base) to 93 +/- 5 bpm (3 mo) (p<0.05)), increased respiratory rate at rest (28+/-5 (base) to 38 +/- 7 (3 mo) (p<0.05)) and increased body weight 77 +/- 2 kg to 81 +/- 2 kg (p<0.05) due to pleural effusion, peripheral edema and ascites. Echocardiographic evaluation revealed significantly an increase of left ventricular enddiastolic diameter from 46 +/- 3 mm (base) to 61 +/- 4 mm (3 mo) (p<0.05). Clinically and echocardiographically no significant changes were revealed in healthy control animals. CONCLUSIONS: We conclude that multiple sequential intracoronary microembolization can effectively induce myocardial dysfunction with clinical and echocardiographical signs of chronic ischemic cardiomyopathy. The present model may be suitable in experimental work on heart failure and left ventricular assist devices, e.g. for studying the impact of mechanical unloading, mechanisms of recovery and reverse remodeling.

Influence of angiotensin-I-converting-enzyme insertion/deletion gene polymorphism on perioperative hemodynamics after coronary bypass graft surgery.

AIM: The angiotensin I-converting enzyme insertion/ deletion polymorphism (ACE-I/D), including three genotypes (II, ID, DD), with a known impact on midterm mortality and morbidity in patients after coronary artery bypass graft surgery (CABG), was studied. Since this polymorphism has been linked with increased vascular response to phenylephrine during cardiopulmonary bypass (CPB), we investigated its possible effect on perioperative hemodynamics in patients undergoing CABG. METHODS: Genotyping for the ACE-I/D was performed by polymerase chain reaction (PRC) amplification in 110 patients who underwent elective CABG with CPB. Patients were assigned to two groups according to their genotype (group II [II genotype] and group ID/DD [ID and DD genotypes]). Systemic hemodynamics were measured directly before and at 4 h, 9 h, and 19 h after CPB. RESULTS: Genotype distribution of ACE-I/D was 18%, 57%, and 25% in genotypes II, ID, and DD, respectively. The two groups were similar in age (group II: 66+/-6 years, group ID/DD: 66+/-8 years), body-mass-index (BMI) (group II: 28+/-2, group ID/DD: 29+/-5 kg/m2), male: female ratio (group II: 16: 4, group ID/DD: 63: 27) and Euroscore (group II: 3.1+/-1.9, group ID/DD: 3.5+/-2.1). There were no differences in mortality rate or perioperative systemic hemodynamics. The pulmonary vascular resistance before cardiopulmonary bypass was higher in the ID/DD genotypes than in the II genotypes (227+/-121 vs 297+/-169 dyn.s(-1).m2.cm(-5)). Four hours after CPB no difference remained; at 9 h after cardiopulmonary bypass there was a slight difference in pulmonary vascular resistance between the two groups (247+/-134 vs 290+/-117 dyn.s(-1).m2.cm(-5)) and a significant difference in pulmonary arterial pressure (19+/-6 vs 23+/-8); at 19 h after CPB the differences were no longer detectable. CONCLUSION: ACE-I/D had no influence on perioperative systemic hemodynamics. However, transitory differences in pulmonary hemodynamic were observed after CPB. These differences may have been due to changes in serum ACE activity during CPB.

Alterations in the natriuretic hormone system related to cardiopulmonary bypass in infants with congestive heart failure.

This study examined changes in the natriuretic hormone system in five infants with congestive heart failure (CHF) due to intracardiac left-to-right shunting who were exposed to cardiopulmonary bypass (CPB) during surgical repair. Plasma concentrations of three hormones [atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and dendroaspis natriuretic peptide (DNP)] and their secondary messenger, guanosine 3',5'-monophosphate (cGMP), were measured, and the biological activity of the system was quantified. At baseline, BNP and DNP concentrations were normal in our patients, a finding that is strikingly different from that of adult CHF patients, whereas ANP concentrations were elevated. Following CPB, ANP concentrations decreased (median, 175 vs 44 pg/ml; p = 0.043) and BNP concentrations increased (median, 25 vs 66 pg/ ml; p = 0.043), whereas DNP concentrations did not change. Following modified ultrafiltration, BNP concentrations increased (p = 0.043), but other natriuretic peptide concentrations did not change. The calculated biological activity of the natriuretic hormone system decreased following CPB [molar ratio, cGMP / (ANP + BNP + DNP); median, 213 vs 127; p = 0.043)]. Additional studies are needed to expand on these findings and identify patients with other types of congenital heart disease who have perioperative disturbances in the natriuretic hormone system and thus might benefit from pharmacologic intervention.

Procalcitonin is a valuable prognostic marker in cardiac surgery but not specific for infection.

BACKGROUND: The prognostic value of elevated serum levels of procalcitonin (PCT) in patients early after cardiac surgery on cardiopulmonary bypass (CPB) remains unclear. In a prospective study, we investigated whether PCT is useful as a prognostic marker in cardiac surgery with respect to mortality, complications and infections, and whether PCT is a specific marker for occurrence of infections. METHODS: Within 8 months, a subset of 80 high-risk patients (APACHE II-score: 25.1 +/- 4.7 (mean +/- SD)) out of a consecutive cohort of 776 patients was investigated. Demographic data, operative data and clinical endpoints (mortality, infection, severe complication) were documented. Serum levels of PCT were analyzed preoperatively and at postoperative day 1. RESULTS: Hospital mortality in this high-risk group was 21.3 %, infections occurred in 33.8 % and complications in 58.8 % of the patients. Preoperative PCT was normal in all patients. Postoperative PCT was increased in non-survivors compared to survivors (34.3 +/- 7.0 ng/ml vs. 15.9 +/- 4.9 ng/ml; p < 0.05), in patients with severe complications (30.3 +/- 6.7 ng/ml vs. 5.5 +/- 1.4 ng/ml; p < 0.05) and in patients with infections (38.4 +/- 11.3 ng/ml vs. 10.8 +/- 1.6 ng/ml; p < 0.05). Area under receiver operating characteristic curve for PCT as predictor of mortality, infections and complications was 0.772 (95 %-confidence-interval (CI): 0.651 - 0.894), 0.720 (95 %-CI: 0.603 - 0.837) and 0.861 (95 %-CI: 0.779 - 0.943), respectively. PCT was not different with infectious compared to non-infectious complications. CONCLUSIONS: High levels of PCT are associated with mortality, infections, and severe complications early after cardiac surgery using cardiopulmonary bypass and therefore provide a valuable prognostic marker. However, PCT does not discriminate between infectious and non-infectious complications.

The St. Jude "Silzone" valve: midterm results in treatment of active endocarditis.

BACKGROUND: The Silzone-coated St. Jude Medical valve (SJM "Silzone" valve), developed to reduce prosthetic valve endocarditis (PVE), was recalled by SJM due to a higher rate of paravalvular leaks. The aim of this study was to determine the efficacy of the SJM "Silzone" valve in avoiding PVE and to evaluate the frequency of paravalvular leaks, when the valve was used exclusively for active bacterial endocarditis. METHODS: From January 1998 to December 1999, the SJM "Silzone" valve was implanted in 40 consecutive patients with active endocarditis (20 aortic, 14 mitral, and 6 both valves). Late transesophageal echocardiography was performed in 87% of survivors, and transthoracic echocardiography in the remaining 13%. Follow-up was 100%. RESULTS: Hospital mortality was 17.5%. Early PVE occurred in 2 of 40 patients (5%). There were two late deaths without signs of recurrent PVE. A hemodynamic relevant paravalvular leak necessitating reoperation was seen in 2 patients within 6 months after operation. The rate of a minor paravalvular leak was 13% (4 of 31 patients). CONCLUSIONS: The SJM "Silzone" valve when implanted for active bacterial endocarditis does not give better results than other mechanical prostheses with regard to early recurrence of endocarditis. The rate of a hemodynamic relevant paravalvular leak requiring reoperation seems rather high during the early postoperative period, whereas the occurrence of minor paravalvular leaks is comparable with that of other mechanical prostheses. Routine observation, recommended for all patients with mechanical heart valves, is also sufficient for patients with the SJM "Silzone" valve.

Aortopexy reduces anastomosis stress after repair of coarctation.

Restenosis after repair of coarctation with hypoplastic distal aortic arch is an important complication. Complete removal of ductal tissue, resection of isthmus area, and side-to-side arterioplasty of the distal aortic arch leads to a wide distance between the aortic arch and descending aorta; therefore, the anastomosis may remain under tension, increasing risk of restenosis. To reduce the tension, aortopexy of the descending aorta was used. The operative technique and the results in 16 neonates and infants are presented.

Combined mitral valve and coronary artery surgery: ischemic versus non-ischemic mitral valve disease.

OBJECTIVES: Mitral valve combined with coronary artery surgery is associated with a higher hospital mortality than each operation in particular. Controversy exists regarding the predictive value of ischemic mitral valve disease (MVD) on outcome. METHODS: Between 1984 and 1997, 262 patients underwent mitral valve operations (replacement, n = 198; repair, n = 64) in combination with coronary revascularization. The etiology of MVD was secondary to ischemic heart disease (group I) in 82 (31%) patients, and non-ischemic (group II) in 180 (69%) patients (rheumatic, 139 patients (53%); degenerative, 41 patients (16%)). Both groups were similar in age, cardiac risk factors and pulmonary artery pressure. Patients of group I had significantly more severe coronary artery disease, more often an impaired left ventricle and myocardial infarction, and were in a worse functional condition. The mean number of bypass grafts was significantly higher in group I. The follow-up was 98% (230/234 patients). RESULTS: With 19.5%, the hospital mortality was significantly increased in group I compared with 6.7% in group II (P = 0.002; overall, 10.7%). Mitral valve repair or replacement had no influence on early outcome, although mitral valve repair was performed more often in group I (37 versus 19%). The survival (valve-related event-free survival) after discharge from hospital in the 1st, 5th and 10th year was 94 (94%), 70 (66%) and 53% (35%) in group I and 96 (95%), 79 (76%) and 54% (41%) in group II, respectively. The long-term functional capacity was equally good in both groups (New York Heart Association mean, 1.86 versus 1.72). CONCLUSIONS: Patients with ischemic MVD are in a worse cardiac condition with significantly higher hospital mortality than patients with non-ischemic MVD and coronary artery bypass grafting. Once discharged from hospital, both groups have comparable long-term outcomes, with the best results in patients with degenerative MVD.

[Risk assessment of mitral valve surgery combined with myocardial revascularization]. / Valoración del riesgo de la cirugía mitral combinada con la revascularización miocárdica.

INTRODUCTION AND OBJECTIVES: Patients with combined mitral valve operation and coronary artery surgery represent a high risk group. The aim of this retrospective study was to evaluate which factors affect early and late postoperative results in this particular group of considered high risk patients. PATIENTS AND METHOD. Between 1984 and 1997, 264 patients (mean age: 63 +/- 7.3 years) underwent mitral valve surgery (199 patients; 75% mitral valve replacement, 25% mitral valve repair) in combination with coronary revascularization (mean 2.4 +/- 1.3 grafts). Follow-up comprised a mean of 69 +/- 42 months and was 98.3% complete. RESULTS: Early mortality was 10.6% (28/264). Ischemic mitral regurgitation operated on in emergent status, moderate to severe reduced left ventricular function and advanced age (> 60 years) were independently associated with early hospital mortality (p < 0.05). Ischemic etiology of mitral valve disease (emergency and elective operations), severity of mitral regurgitation and New York Heart Association (NYHA) functional class IV were related to early hospital mortality, only with univariate statistics. Actuarial survival was 86, 69 and 48% at 1, 5 and 10 years, respectively. The preoperative NYHA functional class was the only variable independently related to late survival. Eighty-five percent of the surviving patients were in NYHA functional class I and II. CONCLUSIONS: Mitral valve operation combined with coronary artery bypass grafting is associated with a high early hospital mortality. Independent risk factors of early mortality are emergency operation of ischemic mitral valve disease, reduced left ventricular function and advanced age. Long term survival is independently influenced only by preoperative NYHA functional class IV.

Missiles in the heart causing coronary artery disease 44 years after injury.

We present a case of two missiles in the heart causing coronary artery disease with first onset 44 years after the injury. The missiles were close to the right coronary artery and the left anterior descending coronary artery. The missiles initiated local arteriosclerosis in these vessels, with an extremely stretched time frame from injury to the onset of symptoms for coronary artery disease.

Surgical therapy of fulminant pulmonary embolism: early and late results.

BACKGROUND: Pulmonary embolectomy remains the only option for patients with fulminant pulmonary embolism and failure or contraindication of thrombolysis even today. Increasing prevalence of heparin-induced thrombocytopenia type II (HIT) adds a new significant problem, which was investigated in a retrospective study. METHODS: Between 1/1979 and 1/1998 41 patients (21 male; age: 51.1 +/- 14.8 years) with fulminant pulmonary embolism underwent pulmonary embolectomy under cardiopulmonary bypass: group I (1979-89): 31 patients; group II (1990-98): 10 patients. Group II included only patients who did not meet the criteria for acute thrombolysis, in 4 patients a HIT was preoperatively assured. All patients were in strongly compromised hemodynamic condition (33/41 high-dose catecholamines, 24/41 mechanical ventilation, 14/41 preoperative cardiopulmonary resuscitation). RESULTS: Perioperative mortality was 29% (group I: 9/31; group II: 3/10; n.s.) Preoperative resuscitation was the only predictive factor (with resuscitation: 9/14; without resuscitation: 3/27; p < 0.001). Severe but not fatal complications occurred in 11 patients: they fully recovered following treatment. Follow-up was completed to 93% (281 patient-years; mean: 10.6 years) and discovered 5 late deaths (late mortality: 1.7%/patient-year; 1 patient: bleeding due to anticoagulation; 4 patients: not related to operation). 26/28 (93%) patients were in NYHA functional class I or II. No recurrent pulmonary embolism or late clinical symptoms related to embolectomy were observed. There was no difference between group I and group II (including the 4 patients with HIT) regarding perioperative mortality, complication, and late results. CONCLUSIONS: Pulmonary embolectomy on cardiopulmonary bypass remains an adequate therapy in patients with failure of or contraindication to thrombolysis, and HIT is not a contraindication.

Elevated levels of a U4/U6.U5 snRNP-associated protein, Spp381p, rescue a mutant defective in spliceosome maturation.

U4 snRNA release from the spliceosome occurs through an essential but ill-defined Prp38p-dependent step. Here we report the results of a dosage suppressor screen to identify genes that contribute to PRP38 function. Elevated expression of a previously uncharacterized gene, SPP381, efficiently suppresses the growth and splicing defects of a temperature-sensitive (Ts) mutant prp38-1. This suppression is specific in that enhanced SPP381 expression does not alter the abundance of intronless RNA transcripts or suppress the Ts phenotypes of other prp mutants. Since SPP381 does not suppress a prp38::LEU2 null allele, it is clear that Spp381p assists Prp38p in splicing but does not substitute for it. Yeast SPP381 disruptants are severely growth impaired and accumulate unspliced pre-mRNA. Immune precipitation studies show that, like Prp38p, Spp381p is present in the U4/U6.U5 tri-snRNP particle. Two-hybrid analyses support the view that the carboxyl half of Spp381p directly interacts with the Prp38p protein. A putative PEST proteolysis domain within Spp381p is dispensable for the Spp381p-Prp38p interaction and for prp38-1 suppression but contributes to Spp381p function in splicing. Curiously, in vitro, Spp381p may not be needed for the chemistry of pre-mRNA splicing. Based on the in vivo and in vitro results presented here, we propose that two small acidic proteins without obvious RNA binding domains, Spp381p and Prp38p, act in concert to promote U4/U5.U6 tri-snRNP function in the spliceosome cycle.

U1 snRNA is cleaved by RNase III and processed through an Sm site-dependent pathway.

Core snRNP proteins bind snRNA through the conserved Sm site, PuA(U)n>/=3GPu. While yeast U1 snRNA has three matches to the Sm consensus, the U1 3'-terminal Sm site was found to be both necessary and sufficient for U1 function. Mutation of this site inhibited pre-mRNA splicing, blocked cell division and resulted in the accumulation of two 3'-extended forms of the U1 snRNA. Cells which harbor the Sm site mutation lack mature U1 RNA (U1alpha) but have a minor polyadenylated species, U1gamma, and a prominent, non-polyadenylated species, U1beta. Metabolic depletion of the essential Sm core protein, Smd1p, also resulted in the increased accumulation of U1beta and U1gamma. In vitro, synthetic U1 precursors were cleaved by Rnt1p (RNase III) very near the U1beta 3'-end observed in vivo. We propose that U1beta is an Rnt1p-cleaved intermediate and that U1 maturation to the U1alpha form occurs through an Sm-sensitive step. Interestingly, both U1alpha and a second, much longer RNA, U1straightepsilon, were produced in an rnt1 mutant strain. These results suggest that yeast U1 snRNA processing may progress through Rnt1p-dependent and Rnt1p-independent pathways, both of which require a fun-ctional Sm site for final snRNA maturation.

A nonimmunoglobulin transgene and the endogenous immunoglobulin mu gene are coordinately regulated by alternative RNA processing during B-cell maturation.

The immunoglobulin (Ig) genes have been extensively studied as model systems for developmentally regulated alternative RNA processing. Transcripts from these genes are alternatively processed at their 3' ends to yield a transcript that is either cleaved and polyadenylated at a site within an intron or spliced to remove the poly(A) site and subsequently cleaved and polyadenylated at a downstream site. Results obtained from expressing modified genes in established tissue culture cell lines that represent different stages of B-lymphocyte maturation have suggested that the only requirement for regulation is that a pre-mRNA contain competing cleavage-polyadenylation and splice reactions whose efficiencies are balanced. Since several non-Ig genes modified to have an Ig gene-like structure are regulated in cell lines, Ig-specific sequences are not essential for this control. This strongly implies that changes in the amounts or activities of general RNA processing components mediate the processing regulation. Despite numerous studies in cell lines, this model of Ig gene regulation has never been tested in vivo during normal lymphocyte maturation. We have now introduced a non-Ig gene with an Ig gene-like structure into the mouse germ line and demonstrate that RNA from the transgene is alternatively processed and regulated in murine splenic B cells. This establishes that the balance and arrangement of competing cleavage-polyadenylation reactions are sufficient for RNA processing regulation during normal B-lymphocyte development. These experiments also validate the use of tissue culture cell lines for studies of Ig processing regulation. This is the first transgenic mouse produced to test a specific model for regulated mRNA processing.

The polyadenylation factor CstF-64 regulates alternative processing of IgM heavy chain pre-mRNA during B cell differentiation.

The switch from membrane-bound to secreted-form IgM that occurs during differentiation of B lymphocytes has long been known to involve regulated processing of the heavy chain pre-mRNA. Here, we show that accumulation of one subunit of an essential polyadenylation factor (CstF-64) is specifically repressed in mouse primary B cells and that overexpression of CstF-64 is sufficient to switch heavy chain expression from membrane-bound (microm) to secreted form (micros). We further show that CstF-64 is limiting for formation of intact CstF, that CstF has a higher affinity for the microm poly(A) site than for the micros site, and that the microm site is stronger in a reconstituted in vitro processing reaction. Our results indicate that CstF-64 plays a key role in regulating IgM heavy chain expression during B cell differentiation.