The conserved npl4 protein complex mediates proteasome-dependent membrane-bound transcription factor activation.

Proteolytic activation of membrane-bound transcription factors has emerged as an important mechanism for the regulation of gene expression. Two membrane-bound transcription factors regulated in this manner are the Saccharomyces cerevisiae proteins Mga2p and Spt23p, which direct transcription of the Delta9-fatty acid desaturase gene OLE1. We now show that a membrane-associated complex containing the highly conserved Npl4p, Ufd1p, and Cdc48p proteins mediates the proteasome-regulated cleavage of Mga2p and Spt23p. Mutations in NPL4, UFD1, and CDC48 cause a block in Mga2p and Spt23p processing, with concomitant loss of OLE1 expression. Taken together, our data indicate that the Npl4 complex may serve to target the proteasome to the ubiquitinated endoplasmic reticulum membrane-bound proteins Mga2p and Spt23p. Given the recent finding that NPL4 is allelic to the ERAD gene HRD4, we further propose that this NPL4 function extends to all endoplasmic reticulum-membrane-associated targets of the proteasome.

Messenger RNAs are recruited for nuclear export during transcription.

Following transcription and processing, eukaryotic mRNAs are exported from the nucleus to the cytoplasm for translation. Here we present evidence that mRNAs are targeted for nuclear export cotranscriptionally. Combined mutations in the Saccharomyces cerevisiae hnRNP Npl3 and TATA-binding protein (TBP) block mRNA export, implying that cotranscriptional recruitment of Npl3 is required for efficient export of mRNA. Furthermore, Npl3 can be found in a complex with RNA Pol II, indicating that Npl3 associates with the transcription machinery. Finally, Npl3 is recruited to genes in a transcription dependent manner as determined by chromatin immunoprecipitation. Another mRNA export factor, Yra1, also associates with chromatin cotranscriptionally but appears to be recruited at a later step. Taken together, our results suggest that export factors are recruited to the sites of transcription to promote efficient mRNA export.

Uncoupling of the hnRNP Npl3p from mRNAs during the stress-induced block in mRNA export.

Npl3p, the major mRNA-binding protein of the yeast Saccharomyces cerevisiae shuttles between the nucleus and the cytoplasm. A single amino acid change in the carboxyl terminus of Npl3p (E409 --> K) renders the mutant protein largely cytoplasmic because of a delay in its import into the nucleus. This import defect can be reversed by increasing the intracellular concentration of Mtr10p, the nuclear import receptor for Npl3p. Conversely, using this mutant, we show that Npl3p and mRNA export out of the nucleus is significantly slowed in cells bearing mutations in XPO1/CRM1, which encodes the export receptor for NES-containing proteins and in RAT7, which encodes an essential nucleoporin. Interestingly, following induction of stress by heat shock, high salt, or ethanol, conditions under which most mRNA export is blocked, Npl3p is still exported from the nucleus. The stress-induced export of Npl3p is independent of both the activity of Xpo1p and the continued selective export of heat-shock mRNAs that occurs following stress. UV-cross-linking experiments show that Npl3p is bound to mRNA under normal conditions, but is no longer RNA associated in stressed cells. Taken together, we suggest that the uncoupling of Npl3p and possibly other mRNA-binding proteins from mRNAs in the nucleus provides a general switch that regulates mRNA export. By this model, under normal conditions Npl3p is a major component of an export-competent RNP complex. However, under conditions of stress, Npl3p no longer associates with the export complex, rendering it export incompetent and thus nuclear.

A member of the Ran-binding protein family, Yrb2p, is involved in nuclear protein export.

Yeast cells mutated in YRB2, which encodes a nuclear protein with similarity to other Ran-binding proteins, fail to export nuclear export signal (NES)-containing proteins including HIV Rev out of the nucleus. Unlike Xpo1p/Crm1p/exportin, an NES receptor, Yrb2p does not shuttle between the nucleus and the cytoplasm but instead remains inside the nucleus. However, by both biochemical and genetic criteria, Yrb2p interacts with Xpo1p and not with other members of the importin/karyopherin beta superfamily. Moreover, the Yrb2p region containing nucleoporin-like FG repeats is important for NES-mediated protein export. Taken together, these data suggest that Yrb2p acts inside the nucleus to mediate the action of Xpo1p in at least one of several nuclear export pathways.

Ubiquitous expression and testis-specific alternative polyadenylation of mRNA for the human Ran GTPase activator RanGAP1.

RanGAP1 is the activator of the Ras-related nuclear GTPase Ran, which is involved in the nucleo-cytoplasmic transport of both, proteins and mRNAs, and also in cell cycle regulation. Here, we report a 2970-bp cDNA clone of RanGAP1 isolated from a HeLa lambda gt11 cDNA library. It contains a 215-bp 5' untranslated region (UTR) with a G + C-content of 68%, followed by a 1764-bp open reading frame and a 989-bp 3' UTR preceding a 77-bp poly(A)+ tail. RanGAP1 shows differential patterns of expression in human tissues. In addition to the 3.5-kb transcript present in all tissues and highly expressed in brain, thymus and testis, we found a second transcript of 2.8 kb in testis. In order to analyze this shorter transcript, we screened a human testis lambda gt10 cDNA library and cloned an alternatively polyadenylated RanGAP1 transcript. Taking the 3' UTR of RanGAP1, which lies downstream of the first polyadenylation signal, as a probe in Northern blot analysis, we confirmed that this second transcript found in testis results from a distinct 3' UTR.

Co-activation of RanGTPase and inhibition of GTP dissociation by Ran-GTP binding protein RanBP1.

RCC1 (the regulator of chromosome condensation) stimulates guanine nucleotide dissociation on the Ras-related nuclear protein Ran. Both polypeptides are components of a regulatory pathway that has been implicated in regulating DNA replication, onset of and exit from mitosis, mRNA processing and transport, and import of proteins into the nucleus. In a search for further members of the RCC1-Ran signal pathway, we have identified proteins of 23, 45 and 300 kDa which tightly bind to Ran-GTP but not Ran-GDP. The purified soluble 23 kDa Ran binding protein RanBP1 does not activate RanGTPase, but increases GTP hydrolysis induced by the RanGTPase-activating protein RanGAP1 by an order of magnitude. In the absence of RanGAP, it strongly inhibits RCC1-induced exchange of Ran-bound GTP. In addition, it forms a stable complex with nucleotide-free RCC1-Ran. With these properties, it differs markedly from guanine diphosphate dissociation inhibitors which preferentially prevent the exchange of protein-bound GDP and in some cases were shown to inhibit GAP-induced GTP hydrolysis. RanBP1 is the first member of a new class of proteins regulating the binding and hydrolysis of GTP by Ras-related proteins.

Human RanGTPase-activating protein RanGAP1 is a homologue of yeast Rna1p involved in mRNA processing and transport.

RanGAP1 is the GTPase activator for the nuclear Ras-related regulatory protein Ran, converting it to the putatively inactive GDP-bound state. Here, we report the amino acid sequence of RanGAP1, derived from cDNA and peptide sequences. We found it to be homologous to murine Fug1, implicated in early embryonic development, and to Rna1p from Saccharomyces cerevisiae and Schizosaccharomyces pombe. Mutations of budding yeast RNA1 are known to result in defects in RNA processing and nucleocytoplasmic mRNA transport. Concurrently, we have isolated Rna1p as the major RanGAP activity from Sc. pombe. Both this protein and recombinant Rna1p were found to stimulate RanGTPase activity to an extent almost identical to that of human RanGAP1, indicating the functional significance of the sequence homology. The Ran-specific guanine nucleotide exchange factor RCC1 and its yeast homologues are restricted to the nucleus, while Rna1p is reported to be localized to the cytoplasm. We suggest a model in which both activities, nuclear GDP-to-GTP exchange on Ran and cytoplasmic hydrolysis of Ran-bound GTP, are essential for shuttling of Ran between the two cellular compartments. Thus, a defect in either of the two antagonistic regulators of Ran would result in a shutdown of Ran-dependent transport processes, in agreement with the almost identical phenotypes described for such defects in budding yeast.

Evidence for the existence of a single ubiquitin gene in Giardia lamblia.

All eukaryotes investigated so far contain multiple copies of ubiquitin genes, most of which are arranged in fusions coding for either polyubiquitin or ubiquitin-ribosomal protein constructs; the former are normally under the control of a heat shock promoter. Giardia lamblia, an intestinal parasite, is the most primitive eukaryote known to date. We have investigated the arrangement and expression of ubiquitin genes in this organism by Southern and Northern blotting. Our data strongly suggest that G. lamblia contains just one ubiquitin gene, which consists of a single copy of the coding sequence and the expression of which is not enhanced by heat shock. By pulsed-field gel electrophoresis we localized this gene on the largest of the five giardial chromosomes. These data imply that the ubiquitin system in Giardia has probably been trapped at an original stage.

[Floating intracardiac metastasis]. / Flottierende intrakardiale Metastase.

Two years after the removal of an embryonic testicular tumour from a now 26-year-old man, his general health deteriorated and a holosystolic murmur was heard over the tricuspid valve area. A chest X-ray film demonstrated lung metastases. Biochemical tests were within normal limits, but the human chorionic gonadotrophin level was elevated to 81,030 mU/ml. Echocardiography revealed mild tricuspid regurgitation and a floating mass between the right atrium and ventricle, in contact with the tricuspid valve. The described findings did not support the diagnosis of embolus or inflammatory vegetation. Because of the danger of embolization and obstruction of the pulmonary outflow tract the mass was removed surgically. Enzyme-histological examination identified the tissue as a metastasis of the testicular tumour. The patient's condition at first improved, but he soon died of bleeding from the lung which was infiltrated by trophoblast-containing metastases of the testicular tumour.

[Results of emergency bypass operation following percutaneous transluminal coronary angioplasty]. / Ergebnisse der notfallmässigen Bypass-Operation nach perkutaner transluminaler Koronarangioplastie.

Between 1980 and 1988, percutaneous transluminal coronary angioplasty (PTCA) was performed in 1,514 patients. Fifty-five patients (3.6%) underwent emergency coronary bypass surgery because of an acute occlusion of the vessel or a dissection with sustained angina and signs of ischemia on the electrocardiogram. Twenty-five of these 55 patients had a myocardial infarction and 5 patients died, 3 perioperatively, 2 after hospital discharge. The degree of stenosis of the dilated vessel significantly influenced the incidence of infarction, while left ventricular ejection fraction prior to PTCA significantly influenced mortality. Patients who underwent surgery with an occluded vessel experienced myocardial infarction significantly more often (87%) than patients with a patent vessel (24%). The incidence of infarction was 27% when reperfusion of the vessel occluded during PTCA was achieved with a reperfusion catheter, repeated PTCA or intracoronary lysis. The patients' age, presence of unstable angina, left ventricular ejection fraction prior to PTCA, the dilated vessel, the extent of coronary artery disease, collateralization of the dilated vessel, and the time between the onset of the event necessitating bypass surgery and the beginning of extracorporeal circulation were found to have no influence on the incidence of infarction. Patients who died had a significantly lower ejection fraction before PTCA than survivors and all patients who died had experienced a large perioperative myocardial infarction.

[Central nervous system risk factors in heart surgery]. / Zentralnervöse Risikofaktoren in der Herzchirurgie.

Serious complications involving the central nervous system in the course of cardiac surgical procedures have become rare. Nevertheless, CNS dysfunctions still are observed in a considerable number of patients, exceeding by far the number of those at risk from preoperative neurological hazards. The influence of extracorporeal circulation performance and hypothermia on the physiology of cerebral autoregulation, as well as microembolization events seem to be crucial factors in this context. The resulting regional or global posthypoxic changes in brain metabolism may lead to the manifestation of various neurologic and psychiatric disorders in the postoperative course. These often minor disturbances of CNS function can only be detected regularly and diagnosed correctly in a prospective way by consulting specialists in neurology, psychiatry and psychology, as performed at the Department of Thoracic- and Cardiovascular Surgery of the University Hospital in Hamburg since 1974, and currently in use an international multicenter study. We found postoperative neurological abnormalities in more than 50% of our patients. While irreversible brain damage occurred in only 0.5% of cases, about twothird exhibited transient symptoms that were no longer apparent after 8-10 days postoperatively. Obvious psychopathological symptoms were noted in 10% of cases after surgery, and minor, likewise transient, psychiatric disturbances were seen in up to 50% of patients; 20% suffered from long-lasting psychic problems. The subjective complaints in these cases exceeded the results of objective assessments to a remarkable amount. The discussion focuses on a critical valuation of clinical and supplementary examination techniques and on the potential pathophysiologic mechanisms induced by extracorporal circulation.

Detection and quantification of lipoprotein(a) in the arterial wall of 107 coronary bypass patients.

The aim of this study was to determine the extent of accumulation of lipoprotein(a) [Lp(a)] in human arterial wall and to define its potential role in atherogenesis. Biopsies routinely taken from the ascending aorta of 107 patients undergoing aortocoronary bypass surgery were analyzed for lipid and lipoprotein parameters, which were then correlated to serum values. A significant positive correlation was established between serum Lp(a) and arterial wall apolipoprotein (apo)(a) by enzyme-linked immunosorbent assay. High serum Lp(a) also led to a significant increase of apo B in the arterial wall. No significant correlation was found between apo B in serum and aortic tissue. Apo B was found to be partially linked to apo(a) in the aortic extract. Furthermore, apo(a) was found to be intact, as determined by its molecular weight in sodium dodecyl sulfate electrophoresis. This technique also revealed that the apo(a) isoform pattern of aortic homogenate was comparable to the individual serum pattern. Immunohistochemical methods demonstrated a striking colocalization of apo(a) and apo B in the arterial wall, predominantly located extracellularly. Both proteins were increased in atherosclerotic plaques. With density gradient ultracentrifugation, Lp(a)-like particles could be isolated from plaque tissue. This initial study showed that Lp(a) accumulates in the arterial wall, partly in the form of lipoprotein-like particles, therefore contributing to plaque formation and coronary heart disease.

Alcohol-induced coagulation necrosis in cardiac tissue: a new concept in the surgical management of recurrent ventricular arrhythmias.

Post myocardial infarction recurrent ventricular arrhythmias are usually coupled with depressed left ventricular function. We have until recently employed the most widely used surgical techniques such as endocardial resection, cryosurgery and isolation. After these methods had failed in a patient with an extensive septal focus, it finally became possible to effectively ablate the arrhythmogenic area by injection of 10 ml 95% alcohol subendocardially. The following experimental studies were carried out to investigate the effect of direct injection of alcohol on the myocardicytes. 95% alcohol, injected into the apex of the left ventricle of Wistar rats weighing between 150 and 200 g caused extensive coagulation necrosis 5 minutes after injection, as was revealed by histological examination. The site of injection showed marked scar formation after 6 months. On the basis of these findings, 4 more patients were treated successfully with alcohol, which was directly injected subendocardially without endocardial resection using a long needle. The advantage of this procedure is that resection can be avoided, particularly in critical areas such as the septum and regions around papillary muscles.

'Head and heart'--neurological and psychological reactions to open heart surgery.

Neurological and psychological reactions to open-heart surgery are widely underestimated phenomena and occur in a much higher incidence than one might expect. When analyzed retrospectively, up to 3.8% of patients who underwent cardiac surgery at the Hamburg University Hospital exhibited these reactions, whereas 35%-50% presented with symptoms and signs of perioperative CNS dysfunctions in prospective studies at our department. About the same percentages are detected in prospective studies of the patients' perioperative psychopathology, stating that a great number of cardiac patients exceed the normal range of anxious, tense, and depressive moods in this setting. The consequences of these findings for the patients' quality of life and the impact for the perioperative management of patients undergoing open-heart surgery are discussed. The current prospective studies, preliminary results of which are presented here, are part of an international interdisciplinary study, initiated to bring more light into the complicated relations between ECC-assisted cardiac surgery, anaesthesiology, neurology and psychology.

Improved survival up to four years after early coronary thrombolysis.

The long-term prognosis after thrombolytic therapy in patients with acute myocardial infarction (AMI) is unknown. This question was investigated in a 4-year follow-up study of 227 patients. According to the status of reperfusion at the end of the acute catheterization, the patients were divided into a patent (n = 171) and an occluded (n = 56) group. Both hospital and 4-year mortality rates were significantly reduced in the patent group by 13 and 14%, respectively (p less than or equal to 0.005 for both). Baseline variables known to be important for prognosis did not differ between the 2 groups. Patients with a patent infarct artery who underwent early acute coronary artery bypass grafting (CABG) had a greater survival (p less than 0.10) and better left ventricular function (p less than 0.01) than did patients with a patent infarct artery who did not undergo CABG. This difference was associated with a lower frequency of fatal reinfarction and cardiogenic shock in the CABG group. Thus, survival is improved up to 4 years after successful thrombolysis and appears to be further enhanced by early CABG.