[Quality of life in patients who suffered erectile dysfunction and underwent penile implant surgery in terms of sexual satisfaction of the patient and partner]. / Lebensqualität von Patienten mit hydraulischen penilen Implantaten bei erektiler Dysfunktion in Bezug auf die sexuelle Zufriedenheit des Patienten und seinem/seiner Partner/Partnerin.

BACKGROUND: Erectile dysfunction is a condition that shows a continuously growing prevalence in the male population. The penis prosthesis implant (PPI) qualifies as an effective form of therapy. OBJECTIVES: The aim of this study was to analyze the sexual satisfaction rate and quality of life in patients who had suffered from erectile dysfunction and who were treated with a penile prosthesis. The patient's partners were also surveyed. METHODS: We collected data from patients who underwent surgery in the Center of Excellence for Penile Implants, University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany. Questionnaires with validated scores (EDITS, EDITS Partner) were sent to all patients and their partners via mail. RESULTS: The satisfaction rate in this study was high which shows that the patients and partners are pleased, and the high sexual satisfaction rate led to a higher quality of life. CONCLUSION: The penile prosthesis implantation as a last option of therapy for erectile dysfunction is useful and brings more than adequate results.

High-submuscular vs. space of Retzius reservoir placement during implantation of inflatable penile implants.

We have evaluated the data of patients who underwent ectopic high submuscular reservoir placement during implantation of inflatable penile prostheses and compared them to those of patients who underwent to traditional reservoir placement in the space of Retzius (SR). The main focus was on evaluating complications and patient satisfaction rates in both methods of RP. One hundred and forty-two patients underwent implantation of the Coloplast Titan OTR prosthesis with exclusive use of the "Clover Leaf" reservoir. We performed a retrospective evaluation, analyzing the treatment-associated complications by means of the Clavien-Dindo classification. All patients as well as their partners received questionnaires with validated scores. Group I: 70 (49.3%) patients who underwent HSM RP. Group II: 72 (50.7%) patients who underwent SR RP. Neither grade IV nor grade V Clavien-Dindo complications were documented. In total, we observed 4 (3.3%) cases grade III b complications, which resulted in revision. Distribution was as follows: infected device (n = 4), scrotal hematoma (n = 2), scrotal seroma (n = 1), and scrotal skin fistula (n = 1). 88% of the patients with ectopic HSM RP and 81% with traditional SR RP were satisfied with their implant. Of the patients with HSM RP, 64.3% (n = 45; BMI range: 18.5-28.8) reported that they were able to feel their reservoir by palpation immediately after surgery. Palpability disappeared in 80% of the patients in this group (BMI > 26.5) after capsule formation at 3 months post-surgery. Only one patient (1.4%; BMI 20.5) reported that he was able to see the reservoir. Our findings suggest that the novel reservoir placement is safe, efficient and results in excellent patient satisfaction, even if the reservoir is initially palpable or visible.

Identification of RTF1, a novel gene important for TATA site selection by TATA box-binding protein in Saccharomyces cerevisiae.

Interaction of the TATA box-binding protein (TBP) with promoters of RNA polymerase II-transcribed genes is an early and essential step in mRNA synthesis. Previous studies have demonstrated that the rate-limiting binding of TBP to a TATA element can be influenced by transcriptional regulatory proteins. To identify additional factors that may regulate DNA binding by TBP in vivo, we performed a genetic selection for extragenic suppressors of a yeast TBP mutant that exhibits altered and relaxed DNA binding specificity. This analysis has led to the discovery of a previously unidentified gene, RTF1. The original rtf1 suppressor mutation, which encodes a single amino acid change in Rtf1, and an rtf1 null allele suppress the effects of the TBP specificity mutant by altering transcription initiation. Differences in the patterns of transcription initiation in these strains strongly suggest that the rtf1 missense mutation is distinct from a simple loss-of-function allele. The results of genetic crosses indicate that suppression of TBP mutants by mutations in RTF1 occurs in an allele-specific fashion. In a strain containing wild-type TBP, the rtf1 null mutation suppresses the transcriptional effects of a Ty delta insertion mutation in the promoter of the HIS4 gene, a phenotype also conferred by the TBP altered-specificity mutant. Finally, as shown by indirect immunofluorescence experiments, Rtf1 is a nuclear protein. Taken together, our findings suggest that Rtf1 either directly or indirectly regulates the DNA binding properties of TBP and, consequently, the relative activities of different TATA elements in vivo.

Biochemical and genetic characterization of a yeast TFIID mutant that alters transcription in vivo and DNA binding in vitro.

A mutation in the gene that encodes Saccharomyces cerevisiae TFIID (SPT15), which was isolated in a selection for mutations that alter transcription in vivo, changes a single amino acid in a highly conserved region of the second direct repeat in TFIID. Among eight independent spt15 mutations, seven cause this same amino acid change, Leu-205 to Phe. The mutant TFIID protein (L205F) binds with greater affinity than that of wild-type TFIID to at least two nonconsensus TATA sites in vitro, showing that the mutant protein has altered DNA binding specificity. Site-directed mutations that change Leu-205 to five different amino acids cause five different phenotypes, demonstrating the importance of this amino acid in vivo. Virtually identical phenotypes were observed when the same amino acid changes were made at the analogous position, Leu-114, in the first repeat of TFIID. Analysis of these mutations and additional mutations in the most conserved regions of the repeats, in conjunction with our DNA binding results, suggests that these regions of the repeats play equivalent roles in TFIID function, possibly in TATA box recognition.

Equivalent mutations in the two repeats of yeast TATA-binding protein confer distinct TATA recognition specificities.

To investigate the process of TATA box recognition by the TATA box-binding protein (TBP), we have performed a detailed genetic and biochemical analysis of two Saccharomyces cerevisiae TBP mutants with altered DNA-binding specificity. The mutant proteins have amino acid substitutions (Leu-205 to Phe and Leu-114 to Phe) at equivalent positions within the two repeats of TBP that are involved in TATA element binding. In an in vivo assay that employs a nearly complete set of single point mutations of the consensus TATAAA sequence, one of the TBP mutants (TBP-L114F) recognizes the sequence TATAAG, while the other TBP mutant (TBP-L205F) recognizes one substitution at the first position of the TATA element, CATAAA, and three substitutions at the 3' end of the TATA box. Specificity patterns determined from in vitro transcription experiments with purified recombinant wild-type TBP and TBP-L205F agree closely with those observed in vivo, indicating that altered TATA utilization in the mutant strains is a direct consequence of altered TATA recognition by the mutant TBPs. The distinct TATA recognition patterns exhibited by TBP-L114F and TBP-L205F strongly suggest that in vivo, TBP binds to the TATA element in a specific orientation. The orientation predicted from these studies is further supported by the identification of intragenic suppressors that correct the defect of TBP-L205F. This orientation is consistent with that observed in vitro by crystallographic analyses of TBP-TATA box complexes. Finally, the importance of altered DNA-binding specificity in transcriptional regulation at the S. cerevisiae his4-912 delta promoter was addressed for TBP-L205F. A mutational analysis of this promoter region demonstrates that the nonconsensus TATA element CATAAA is required for a transcriptional effect of TBP-L205F in vivo. This finding suggests that the interaction of TBP with nonconsensus TATA elements may play an important regulatory role in transcription initiation.

Analysis of TFIIA function In vivo: evidence for a role in TATA-binding protein recruitment and gene-specific activation.

Activation of transcription can occur by the facilitated recruitment of TFIID to promoters by gene-specific activators. To investigate the role of TFIIA in TFIID recruitment in vivo, we exploited a class of yeast TATA-binding protein (TBP) mutants that is activation and DNA binding defective. We found that co-overexpression of TOA1 and TOA2, the genes that encode yeast TFIIA, overcomes the activation defects caused by the TBP mutants. Using a genetic screen, we isolated a new class of TFIIA mutants and identified three regions on TFIIA that are likely to be involved in TBP recruitment or stabilization of the TBP-TATA complex in vivo. Amino acid replacements in only one of these regions enhance TFIIA-TBP-DNA complex formation in vitro, suggesting that the other regions are involved in regulatory interactions. To determine the relative importance of TFIIA in the regulation of different genes, we constructed yeast strains to conditionally deplete TFIIA levels prior to gene activation. While the activation of certain genes, such as INO1, was dramatically impaired by TFIIA depletion, activation of other genes, such as CUP1, was unaffected. These data suggest that TFIIA facilitates DNA binding by TBP in vivo, that TFIIA may be regulated by factors that target distinct regions of the protein, and that promoters vary significantly in the degree to which they require TFIIA for activation.

Inhibition of acetyl coenzyme A carboxylase activity restores expression of the INO1 gene in a snf1 mutant strain of Saccharomyces cerevisiae.

Mutations in the Saccharomyces cerevisiae SNF1 gene affect a number of cellular processes, including the expression of genes involved in carbon source utilization and phospholipid biosynthesis. To identify targets of the Snf1 kinase that modulate expression of INO1, a gene required for an early, rate-limiting step in phospholipid biosynthesis, we performed a genetic selection for suppressors of the inositol auxotrophy of snf1Delta strains. We identified mutations in ACC1 and FAS1, two genes important for fatty acid biosynthesis in yeast; ACC1 encodes acetyl coenzyme A carboxylase (Acc1), and FAS1 encodes the beta subunit of fatty acid synthase. Acc1 was shown previously to be phosphorylated and inactivated by Snf1. Here we show that snf1Delta strains with increased Acc1 activity exhibit decreased INO1 transcription. Strains carrying the ACC1 suppressor mutation have reduced Acc1 activity in vitro and in vivo, as revealed by enzymatic assays and increased sensitivity to the Acc1-specific inhibitor soraphen A. Moreover, a reduction in Acc1 activity, caused by addition of soraphen A, provision of exogenous fatty acid, or conditional expression of ACC1, suppresses the inositol auxotrophy of snf1Delta strains. Together, these findings indicate that the inositol auxotrophy of snf1Delta strains arises in part from elevated Acc1 activity and that a reduction in this activity restores INO1 expression in these strains. These results reveal a Snf1-dependent connection between fatty acid production and phospholipid biosynthesis, identify Acc1 as a Snf1 target important for INO1 transcription, and suggest models in which metabolites that are generated or utilized during fatty acid biosynthesis can significantly influence gene expression in yeast.

An in vivo library-versus-library selection of optimized protein-protein interactions.

We describe a rapid and efficient in vivo library-versus-library screening strategy for identifying optimally interacting pairs of heterodimerizing polypeptides. Two leucine zipper libraries, semi-randomized at the positions adjacent to the hydrophobic core, were genetically fused to either one of two designed fragments of the enzyme murine dihydrofolate reductase (mDHFR), and cotransformed into Escherichia coli. Interaction between the library polypeptides reconstituted enzymatic activity of mDHFR, allowing bacterial growth. Analysis of the resulting colonies revealed important biases in the zipper sequences relative to the original libraries, which are consistent with selection for stable, heterodimerizing pairs. Using more weakly associating mDHFR fragments, we increased the stringency of selection. We enriched the best-performing leucine zipper pairs by multiple passaging of the pooled, selected colonies in liquid culture, as the best pairs allowed for better bacterial propagation. This competitive growth allowed small differences among the pairs to be amplified, and different sequence positions were enriched at different rates. We applied these selection processes to a library-versus-library sample of 2.0 x 10(6) combinations and selected a novel leucine zipper pair that may be appropriate for use in further in vivo heterodimerization strategies.

Transcription termination in Escherichia coli. Measurement of the rate of enzyme release from Rho-independent terminators.

The termination/release phase of transcription must involve at least three major steps: cessation of elongation; release of the transcript; and release of the RNA polymerase. We have devised a novel method for measuring the rate of Escherichia coli RNA polymerase release during transcription termination. The method is based on a kinetic analysis of the rate of RNA synthesis during steady-state transcription. Using this method with defined transcription units, we have found that RNA polymerase release occurs rapidly from several rho-independent terminators. Enzyme release from the T7 early terminator occurs within 13(+/- 3) seconds of the cessation of elongation. Neither nusA protein nor supercoiling of the DNA template affects the rate of enzyme release. However, addition of excess sigma factor significantly increases the rate of enzyme recycling during the steady state. Since added sigma factor does not alter the rates of initiation and elongation by E. coli RNA polymerase holoenzyme, it appears that sigma factor stimulates one or more steps in the termination/release process and reduces the rate of enzyme release to a few seconds. We present evidence that suggests sigma may be directly involved in catalyzing release of the core RNA polymerase from the DNA template during transcription termination. The rapid rates of enzyme release we measure make it difficult to be certain of the exact pathway of events that occur in the termination/release phase of transcription. The most plausible pathway involves initial release of the RNA transcript followed by release of core RNA polymerase from the DNA. Studies on the properties of core polymerase-RNA complexes indicate that core polymerase and the RNA transcript probably do not dissociate as a complex from the terminator. Furthermore, these core-RNA complexes are too stable to represent significant intermediates in the termination/release pathway, at least in the early steps of the reaction.

RNA chain elongation by Escherichia coli RNA polymerase. Factors affecting the stability of elongating ternary complexes.

We have devised a method to follow the stability of individual ternary transcription complexes containing Escherichia coli RNA polymerase halted at many different sites along a DNA template during the transcription process. Studies of complexes formed with phage T7 DNA templates reveal at least three general classes of ternary complexes that differ dramatically in their properties. Complexes of one sort (normal complexes) are highly stable to dissociation and denaturation under a variety of solution conditions. They remain intact and active for up to 24 hours even in salt concentrations up to 1 M-K+. This suggests that they are stabilized to a significant extent by non-ionic interactions between RNA polymerase and the nucleic acids. We consider these to be the normal complexes formed during RNA chain elongation. Complexes of a second sort (release complexes) dissociate rapidly, releasing free RNA transcripts and active RNA polymerase. The rate of dissociation is substantially enhanced by elevated concentrations of K+, hence the interaction between RNA polymerase and nucleic acids in these complexes is stabilized predominantly by ionic interactions. However, release complexes are stabilized by millimolar concentrations of Mg2+, which as been implicated in stabilization of the binding of RNA to free RNA polymerase. These complexes are formed at DNA sequences that we refer to as release sites. Analysis of DNA sequences at release sites reveals that all share a common feature, the potential to form an RNA hairpin in the region just upstream from the actual 3' end of the released RNA. Experiments incorporating IMP in the transcript and blocking potential hairpin formation with DNA oligomers support a direct role for an RNA hairpin in triggering the release reaction. Changes in the 3'-proximal DNA sequences generally have little effect on the presence or rate of the release reaction, although there are significant exceptions. The results suggest that the presence of certain RNA hairpins in the region six to ten nucleotides upstream from the transcript growing point can trigger a substantial structural transition in the ternary transcription complex, forming a "release mode" complex from which transcript dissociation is facilitated. This release, mode complex may be a central intermediate in RNA chain termination. A final class of complexes (dead-end complexes) appear to be elongating complexes that have entered a state or conformation that is stable, but is blocked in resuming the normal elongation reaction. Such complexes bear active RNA polymerase, and can be restarted after limited pyrophosphorolysis. The structural elements that determine the formation of dead-end complexes are not yet known.

Helix-stabilized Fv (hsFv) antibody fragments: substituting the constant domains of a Fab fragment for a heterodimeric coiled-coil domain.

Antibody Fv fragments would in principle be useful for a variety of biotechnological applications because of their small size and the possibility to produce them in relatively large amounts in recombinant form; however, their limited stability is a drawback. To solve this problem, both domains are usually fused via a peptide linker to form a single-chain Fv (scFv) fragment, but in some cases this leads to a dimerization. We present an alternative format for stabilizing antibody Fv fragments. The C(H)1 and C(L) domain of the Fab fragment were replaced with a heterodimeric coiled coil (WinZip-A2B1), which had previously been selected using a protein-fragment complementation assay in Escherichia coli. This new antibody format was termed helix-stabilized Fv fragment (hsFv), and was compared to the corresponding Fv, Fab and single-chain Fv format. Bacterial growth and expression of the hsFv was significantly improved compared to the Fab fragment. The hsFv fragment formed a heterodimer of heavy and light chain with the expected molecular mass, also under conditions where the scFv fragment was predominantly dimeric. The hsFv fragment was significantly more stable than the Fv fragment, and nearly as stable as the scFv fragment under the conditions used (80 nM protein concentration). Thus, the format of a helix-stabilized Fv (hsFv) fragment can be a useful alternative to existing recombinant antibody formats, especially in cases where poor expression of Fab fragments or multimerization of scFv fragments is a problem.

A heterodimeric coiled-coil peptide pair selected in vivo from a designed library-versus-library ensemble.

Novel heterodimeric coiled-coil pairs were selected simultaneously from two DNA libraries using an in vivo protein-fragment complementation assay with dihydrofolate reductase, and the best pair was biophysically characterized. We randomized the interface-flanking e and g positions to Gln, Glu, Arg or Lys, and the core a position to Asn or Val in both helices simultaneously, using trinucleotide codons in DNA synthesis. Selection cycles with three different stringencies yielded sets of coiled-coil pairs, of which 80 clones were statistically analyzed. Thereby, properties most crucial for successful heterodimerization could be distinguished from those mediating more subtle optimization. A strong bias towards an Asn pair in the core a position indicated selection for structural uniqueness, and a reduction of charge repulsions at the e/g positions indicated selection for stability. Increased stringency led to additional selection for heterospecificity by destabilizing the respective homodimers. Interestingly, the best heterodimers did not contain exclusively complementary charges. The dominant pair, WinZip-A1B1, proved to be at least as stable in vitro as naturally occurring coiled coils, and was shown to be dimeric and highly heterospecific with a K(D) of approximately 24 nM. As a result of having been selected in vivo it possesses all characteristics required for a general in vivo heterodimerization module. The combination of rational library design and in vivo selection presented here is a very powerful strategy for protein design, and it can reveal new structural relationships.

Synthetic lethal interactions suggest a role for the Saccharomyces cerevisiae Rtf1 protein in transcription elongation.

Strong evidence indicates that transcription elongation by RNA polymerase II (pol II) is a highly regulated process. Here we present genetic results that indicate a role for the Saccharomyces cerevisiae Rtf1 protein in transcription elongation. A screen for synthetic lethal mutations was carried out with an rtf1 deletion mutation to identify factors that interact with Rtf1 or regulate the same process as Rtf1. The screen uncovered mutations in SRB5, CTK1, FCP1, and POB3. These genes encode an Srb/mediator component, a CTD kinase, a CTD phosphatase, and a protein involved in the regulation of transcription by chromatin structure, respectively. All of these gene products have been directly or indirectly implicated in transcription elongation, indicating that Rtf1 may also regulate this process. In support of this view, we show that RTF1 functionally interacts with genes that encode known elongation factors, including SPT4, SPT5, SPT16, and PPR2. We also show that a deletion of RTF1 causes sensitivity to 6-azauracil and mycophenolic acid, phenotypes correlated with a transcription elongation defect. Collectively, our results suggest that Rtf1 may function as a novel transcription elongation factor in yeast.

Evidence for the involvement of the Glc7-Reg1 phosphatase and the Snf1-Snf4 kinase in the regulation of INO1 transcription in Saccharomyces cerevisiae.

Binding of the TATA-binding protein (TBP) to the promoter is a pivotal step in RNA polymerase II transcription. To identify factors that regulate TBP, we selected for suppressors of a TBP mutant that exhibits promoter-specific defects in activated transcription in vivo and severely reduced affinity for TATA boxes in vitro. Dominant mutations in SNF4 and recessive mutations in REG1, OPI1, and RTF2 were isolated that specifically suppress the inositol auxotrophy of the TBP mutant strains. OPI1 encodes a repressor of INO1 transcription. REG1 and SNF4 encode regulators of the Glc7 phosphatase and Snf1 kinase, respectively, and have well-studied roles in glucose repression. In two-hybrid assays, one SNF4 mutation enhances the interaction between Snf4 and Snf1. Suppression of the TBP mutant by our reg1 and SNF4 mutations appears unrelated to glucose repression, since these mutations do not alleviate repression of SUC2, and glucose levels have little effect on INO1 transcription. Moreover, mutations in TUP1, SSN6, and GLC7, but not HXK2 and MIG1, can cause suppression. Our data suggest that association of TBP with the TATA box may be regulated, directly or indirectly, by a substrate of Snf1. Analysis of INO1 transcription in various mutant strains suggests that this substrate is distinct from Opi1.

A dimeric bispecific miniantibody combines two specificities with avidity.

Bispecific antibodies extend the capabilities of nature and might be applied in immunotherapy and biotechnology. By fusing the gene of a single-chain Fv (scFv) fragment to a helical dimerization domain, followed by a second scFv fragment of different specificity, we were able to express a functional protein in E. coli, which is bispecific and has two valencies for each specificity. The dimeric bispecific (DiBi) miniantibody preserves the natural avidity of antibodies in a very small-sized molecule of only 120 kDa. The generality of the principle was shown with a scFv fragment binding the EGF-receptor (named scFv 425) in three combinations with scFv fragments either directed against CD2 (ACID2.M1), phosphorylcholine (McPC603) or fluorescein (FITC-E2). Binding was analyzed by sandwich surface plasmon resonance biosensor (BIAcore) measurements.

The first constant domain (C(H)1 and C(L)) of an antibody used as heterodimerization domain for bispecific miniantibodies.

Bispecific miniantibodies were constructed by genetically fusing the C(H)1 domain of an IgG1 to the C-terminus of a single-chain Fv fragment (scFv-425), specific for the EGF receptor, and fusing the C(L) domain of a kappa light chain to the C-terminus of a scFv specific for CD2 (scFv-M1). An efficient dicistronic gene arrangement for functional expression in Escherichia coli was constructed. Immunoblots demonstrated correct domain assembly and the formation of the natural C(H)1-C(L) disulfide bridge. Gel filtration confirmed the correct size, sandwich ELISAs demonstrated bispecific functionality, and SPR biosensor measurements determined binding to EGF-R in comparison to bivalent constructs. Bispecific anti-EGF-R/anti-CD2 miniantibodies are candidates for the immunotherapy of cancer.

Selectively infective phage (SIP) technology: scope and limitations.

We review here the selectively infective phage (SIP) technology, a powerful tool for the rapid selection of protein-ligand and peptide-ligand pairs with very high affinities. SIP is highly suitable for discriminating between molecules with subtle stability and folding differences. We discuss the preferred types of applications for this technology and some pitfalls inherent in the in vivo SIP method that have become apparent in its application with highly randomized libraries, as well as some precautions that should be taken in successfully applying this technology.