Epitope tagging: general method for tracking recombinant proteins.

Epitope tagging has provided a useful experimental strategy with widespread applicability. The ample variety of epitope tag systems that have been put to use to date provide a collection of attributes relevant to virtually any experimental system. As a consequence, epitope tagging will continue to be a valuable tool for molecular biologists long into the future.

The major nuclear envelope targeting domain of LAP2 coincides with its lamin binding region but is distinct from its chromatin interaction domain.

LAP2 is an integral protein of the inner nuclear membrane which binds lamins and chromosomes and is suggested to have an important role in nuclear envelope organization. In a previous study we identified an internal 76-amino acid region of LAP2 which is required for stable targeting of the protein to the nuclear envelope. Here, we have mapped the lamin binding region of LAP2 and demonstrate that it coincides with this nuclear envelope targeting domain. In contrast, we found that the portion of LAP2 involved in binding to chromosomes resides in a separate region of the protein near its NH2 terminus. The minimal lamin binding region of LAP2 is capable of conferring stable nuclear envelope localization when attached to the transmembrane and partial lumenal domains of a protein that shows no nuclear envelope targeting activity. This directly supports the notion that a major mechanism for localization of integral membrane proteins at the inner nuclear membrane involves binding to lamins, which would constrain diffusion through the continuous nuclear envelope/endoplasmic reticulum membrane system.

Direct evidence for SIR2 modulation of chromatin structure in yeast rDNA.

The yeast SIR2 gene maintains inactive chromatin domains required for transcriptional repression at the silent mating-type loci and telomeres. We previously demonstrated that SIR2 also acts to repress mitotic and meiotic recombination between the tandem ribosomal RNA gene array (rDNA). Here we address whether rDNA chromatin structure is altered by loss of SIR2 function by in vitro and in vivo assays of sensitivity to micrococcal nuclease and dam methyltransferase, respectively, and present the first chromatin study that maps sites of SIR2 action within the rDNA locus. Control studies at the MAT alpha locus also revealed a previously undetected MNase-sensitive site at the a1-alpha 2 divergent promoter which is protected in sir2 mutant cells by the derepressed a1-alpha 2 regulator. In rDNA, SIR2 is required for a more closed chromatin structure in two regions: SRR1, the major SIR-Responsive Region in the non-transcribed spacer, and SRR2, in the 18S rRNA coding region. None of the changes in rDNA detected in sir2 mutants are due to the presence of the a1-alpha 2 repressor. Reduced recombination in the rDNA correlates with a small, reproducible transcriptional silencing position effect. Deletion and overexpression studies demonstrate that SIR2, but not SIR1, SIR3 or SIR4, is required for this rDNA position effect. Significantly, rDNA transcriptional silencing and rDNA chromatin accessibility respond to SIR2 dosage, indicating that SIR2 is a limiting component required for chromatin modeling in rDNA.

High levels of the GTPase Ran/TC4 relieve the requirement for nuclear protein transport factor 2.

The GTPase Ran/TC4 and the 14-kDa protein nuclear transport factor 2 (NTF2) are two of the cytosolic factors that mediate nuclear protein import in vertebrates. Previous biochemical studies have shown that NTF2 binds directly to the GDP-bound form of Ran/TC4 and to proteins of the nuclear pore complex that contain phenylalanine-glycine repeats. In the present study we have used molecular genetic approaches to study the Saccharomyces cerevisiae homologue of NTF2. The scNTF2 gene encodes a protein that is 44% identical to the human protein. We found that deletion of the scNTF2 gene is lethal and that repression of NTF2p expression by a regulatable promoter results in gross structural distortions of the nuclear envelope. In a screen for high copy number suppressors of a scNTF2 deletion, the only gene we isolated other than scNTF2 itself was GSP1, the S. cerevisiae homologue of Ran/TC4. Furthermore, we found that high levels of Ran/TC4 can relieve the requirement for NTF2 in a mammalian-permeabilized cell assay for nuclear protein import. These data suggest that certain of the nuclear protein import functions of NTF2 and Ran/TC4 are closely linked and that NTF2 may serve to modulate a transport step involving Ran/TC4.

[Endoscopic laser therapy in gastroenterology--physical-technical principles and applications]. / Endoskopische Lasertherapie in der Gastroenterologie--Physikalisch-technische Grundlagen und Applikationen.

Since the mid-seventies the application of laser in gastroenterology for the treatment of haemorrhages and tumours has stood the test. At present the neodym-YAG-laser is the most important endoscopically applicable laser. The sources of laser radiation, the biophysical effects of laser and their fields of application, the technical and methodical and security-technical aspects as well as own divice-technical solutions and the indications in the application of the endoscopic laser therapy are described. In 12 out of 15 patients with a villous adenoma of the rectum the treatment was successful. In 9 patients with a high-degree stenosing rectosigmoid carcinoma (diameter 0.5 cm) the passage could be restored (diameter after treatment 1.3 cm). Complications did not appear. The laser therapy is effective, poor in risks, but not cheap.

[Endoscopic laser treatment of neoplasm and tubulovillous adenoma of the rectosigmoid. Initial experiences and review of the literature]. / Endoskopische Lasertherapie beim Malignom und tubulovillösen Adenom des Rektosigmoids. Erste Erfahrungen und Literaturübersicht.

With laser it's possible to produce a radiation of definite wave length which is rich in energy. This energy is changed into heat during its impinge on tissue. Different effects (coagulation, vaporization) can be produced in dependence on wave length, the technical features of laser and the different interaction of laser light. In 8 patients with obstructive polypous carcinomas of rectosigmoid a complete passage-recovery could be achieved. The therapy was effective in 8 out of 11 patients with villous adenomas. One patient had a relapse into adenoma after half a year. With laser a distal rectostenosis at high degree could be widened at Morbus Crohn. Complications didn't appear. Indications of laser-therapy are explained. The laser-therapy is effective, not very risky but not cheap. Therefore it requires an application of overlapping subjects.

[Primary clinical, endoscopic and histologic findings in solitary rectal ulcer]. / Klinische, endoskopische und histologische Erstbefunde beim solitären Rektumulkus.

This study presents clinical, endoscopical and histological primary findings of solitary ulcer of the rectum obtained in 24 patients. Solitary ulcer of the rectum is preferably a disease of young women. The main symptom is recurrent rectal bleeding. Endoscopically in 50% of all cases mucosal ulcerations can be observed. The other cases are non-ulcerous lesions with uncharacteristic endoscopical findings. The majority of the lesions are within 6-8 cm of the anal margin. In 44% of the patients there are findings, which are suspicious of preexisting rectal prolapse. The histological main criterion is a fibromuscular hyperplasia of the lamina propria. Full agreement of endoscopical and histological diagnosis at primary examination could be obtained in only 17% of the cases.

Polyoma virus DNA replication is semi-discontinuous.

In marked contrast to simian virus 40 (SV40), polyoma virus (PyV) has been reported to replicate discontinuously on both arms of replication forks. In an effort to clarify the relationship between the mechanisms of DNA replication in these closely related viruses, the distribution of RNA-primed DNA chains at replication forks was examined concurrently in PyV and SV40 replicating DNA purified from virus-infected cells. About one third of PyV DNA chains contained 7 to 9 ribonucleotides covalently linked to their 5'-end. A similar fraction of DNA chains from replicating SV40 DNA contained an oligoribonucleotide that was 6 to 9 residues long and began with either (p)ppA or (p)ppG. Greater than 80% of PyV or SV40 RNA-primed DNA chains hybridized specifically to the retrograde template. Moreover, at least 95% of the RNA-primed DNA chains from either PyV or SV40 whose initiation sites could be mapped to unique nucleotide locations originated from the retrograde template. Therefore, PyV and SV40 DNA replication forks are essentially the same; DNA synthesis is discontinuous predominantly, if not exclusively, on the retrograde template.

The origin of bidirectional DNA replication in polyoma virus.

The nucleotide locations of RNA-p-DNA covalent linkages in polyoma virus (PyV) replicating DNA were mapped in the region containing the genetically required origin of DNA replication (ori). These linkages mark the initiation sites for RNA-primed DNA synthesis. A clear transition was identified between the presence of these linkages (discontinuous DNA synthesis) and their absence (continuous DNA synthesis) on each strand of ori. This demonstrated that PyV DNA replication, like simian virus 40 (SV40), is semi-discontinuous, and thus revealed the location of the origin of bidirectional DNA replication (OBR). The transition site on the template encoding PyV late mRNA occurred at the junction of ori-core and T-antigen binding site A. This was essentially the same site as previously observed in SV40 (Hay and DePamphilis, 1982). However, in contrast to SV40, the transition site on the template encoding PyV early mRNA was displaced towards the late gene side of ori. This resulted in a 16 nucleotide gap within ori in which no RNA-p-DNA linkages were observed on either strand. A model for the initiation of PyV DNA replication is presented.

[Causes of gastrointestinal hemorrhages in chronic liver diseases]. / Ursachen gastrointestinaler Blutungen bei chronischen Leberkrankheiten.

356 patients with chronic liver diseases hospitalized in our clinic during the period from January 1, 1981, to December 12, 1983, were enrolled in a retrospective study. Of them 55 had been admitted for acute gastrointestinal hemorrhage. It appears that gastroscopy is the only method allowing to localize the origin of the bleeding with high certainty. Bleedings from varices (45%) and ulcers or erosions (22%) were seen most frequently. With regard to case history, clinic, paraclinic and prognosis, no significant differences were found between these various origins of the hemorrhages. Also it is remarkable that all bleedings occur with higher frequency in portal hypertension.

[Emergency endoscopy in chronic liver diseases]. / Notfallendoskopie bei chronischen Lebererkrankungen.

Emergency endoscopy has to be regarded as a method of high diagnostic value. Based on the resulting therapeutic consequences and with allowance for the prerequisites and contraindications, emergency endoscopy in chronic liver diseases is to be considered a purposeful method. If endoscopic treatment possibilities in bleedings of the gastrointestinal tract continue to improve, the demonstration of an improvement in prognosis by emergency endoscopy seems to be merely a question of time.

[Early stomach cancer in our clinical sample]. / Das Magenfrühkarzinom im eigenen Krankengut.

In the period from 1971 to 1980 we studied 11 650 patients undergoing endoscopy of the stomach. We found in 427 patients a gastric cancer. In 63 patients we found an early cancer, according to 14% of all gastric cancers. The main symptoms were pain and loss of appetite. In 34% of the patients the duration of complaints was more than 1/2 year. In more than 50% the early cancer was located in the distal part of stomach. In 52% we observed the ulcerative lesion of the gastric mucosa. The histological findings were in 52% the intestinal type and in 72% the diffuse type of early gastric cancer. The precise diagnosis of an early cancer based on the histological examination of the gastric wall after gastric resection, with the exception of loopectomy and polypectomy with well-defined diagnosis of an early cancer. By the aid of preventive examination of patients with so-called precancerous condition of the gastric mucosa we see a possibility to increase the detection of early gastric cancer.

[Aortic arch angiography in the preoperative diagnosis of cerebrovascular diseases. Comparison of the conventional technic and intra-arterial DSA]. / Die Aortenbogenangiographie in der präoperativen Diagnostik zerebraler Gefässerkrankungen. Vergleich von konventioneller Technik und intraarterieller DSA.

Demonstration of the aortic arch is necessary in the investigation of cerebrovascular insufficiency for showing the origins and course of the cerebral arteries and for detecting steal syndromes and anatomical variations. Intra-arterial DSA is comparable in this respect with the traditional film technique; it has the advantage of reducing contrast doses by 75% and of reducing film costs and the examination time. Reliable evaluation of the carotid bifurcations is possible in less than half the cases. Selective carotid angiography cannot be replaced, therefore, by this technique. Selective angiography is also necessary for the pre-operative study of the intracranial vessels.

[Results of coloscopic polypectomy in respect of the dignity (author's transl)]. / Ergebnisse der koloskopischen Polypektomie im Hinblick auf die Dignität.

The commonest type of polyps in the large intestine in our series of 506 polypectomies in 262 patients were adenomas (64%) especially tubular adenomas. Severe epithelial atypia being present in 11% of all adenomas and it was three times more discovered in villous adenomas than in tubular forms and with increasing adenomas size. Because severe epithelial atypia could be observed in polyps with diameters less than 5 mm it should be demanded to remove each polyp for an exact histological examination independent of his size. Single forceps biopsies of polyps do not allow the estimation of their further development in view of malignancy.

[Emergency endoscopy]. / Notfallendoskopie.

The possibilities and advantages of the emergency endoscopy of the upper intestinal tract are emphasized. With the help of 475 own examinations from 1971 to 1973 and from 1973 to June 1977 the improvement of the diagnostic gain by instruments with prograde optics is proved. Ulcerous diseases of the stomach and the duodenum are found as most frequent cause of haemorrhage which appear in the oesophagus or in the oesophagogastric transition. With 20% the number of the multiple findings was significantly high, the proof of which is to be regarded as an essential advantage of the emergency endoscopy. But at the same time they also emphasize the necessity not to be satisfied with the proof of a source of haemorrhage. The coloscopic emergency endoscopy is more difficult. Endoscopico-therapeutic possiblities increase the importance of the emergency endoscopy.