The protein kinase C-related kinase PRK2 interacts with the protein tyrosine phosphatase PTP-BL via a novel PDZ domain binding motif

Protein tyrosine phosphatase-basophil like (PTP-BL) is a large non-transmembrane protein tyrosine phosphatase implicated in the modulation of the cytoskeleton. Here we describe a novel interaction of PTP-BL with the protein kinase C-related kinase 2 (PRK2), a serine/threonine kinase regulated by the G-protein rho. This interaction is mediated by the PSD-95, Drosophila discs large, zonula occludens (PDZ)3 domain of PTP-BL and the extreme C-terminus of PRK2 as shown by yeast two-hybrid assays and coimmunoprecipitation experiments from transfected HeLa cells. In particular, we demonstrate that a conserved C-terminal cysteine of PRK2 is indispensable for the interaction with PTP-BL. In HeLa cells we demonstrate colocalization of both proteins in lamellipodia like structures. Interaction of PTP-BL with the rho effector kinase PRK2 gives further evidence for a possible function of PTP-BL in the regulation of the actin cytoskeleton.     

Searching sequence space for high-affinity binding peptides using ribosome display

We present the construction of a synthetic library based on the scaffold of bovine heart fatty acid-binding protein (FABP) with 1.1x10(14) independent members. Ribosome display was applied to select streptavidin-binding peptides in vitro from 2x10(13) molecules of the library each encoding FABP with 15 contiguous random amino acid residues at its N terminus. The selection yielded several different binding peptides. The best binder possessed a dissociation constant as low as 4nM and, in contrast to the previously isolated peptides, contained no HPQ motif. A substitution analysis enabled shortening of the 15-mer peptide and revealed a 9-mer variant with a dissociation constant of 17nM, which is a 1000-fold increase of affinity compared to the already known peptides of this size. This high-affinity binding peptide in combination with the whole set of streptavidin conjugates should be an extremely useful tool for the detection and purification of recombinant proteins.     

Essential role of the apolipoprotein E receptor-2 in sperm development

The apolipoprotein (apo) E receptor-2 (apoER2) is a member of the low density lipoprotein receptor gene family and an important regulator of neuronal migration. It acts as a receptor for the signaling factor Reelin and provides positional cues to neurons that migrate to their proper position in the developing brain. Besides brain formation defects, apoER2-deficient mice also exhibit male infertility. The role of the receptor in male reproduction, however, remained unclear. Here we demonstrate that apoER2 is highly expressed in the initial segment of the epididymis, where it affects the functional expression of clusterin and phospholipid hydroperoxide glutathione peroxidase (PHGPx), two proteins required for sperm maturation. Reduced PHGPx expression in apoER2 knockout mice results in the inability of the sperm to regulate the cell volume and in abnormal sperm morphology and immotility. Because insufficient expression of PHGPx is a major cause of infertility in men, these findings not only highlight an important new function for apoER2 that is unrelated to neuronal migration, but they also suggest a possible role for apoER2 in human infertility.     

The concept of fillet flaps: classification, indications, and analysis of their clinical value

Tissue of amputated or nonsalvageable limbs may be used for reconstruction of complex defects resulting from tumor and trauma. This is the "spare parts" concept.By definition, fillet flaps are axial-pattern flaps that can function as composite-tissue transfers. They can be used as pedicled or free flaps and are a beneficial reconstruction strategy for major defects, provided there is tissue available adjacent to these defects.From 1988 to 1999, 104 fillet flap procedures were performed on 94 patients (50 pedicled finger and toe fillets, 36 pedicled limb fillets, and 18 free microsurgical fillet flaps).Nineteen pedicled finger fillets were used for defects of the dorsum or volar aspect of the hand, and 14 digital defects and 11 defects of the forefoot were covered with pedicled fillets from adjacent toes and fingers. The average size of the defects was 23 cm2. Fourteen fingers were salvaged. Eleven ray amputations, two extended procedures for coverage of the hand, and nine forefoot amputations were prevented. In four cases, a partial or total necrosis of a fillet flap occurred (one patient with diabetic vascular disease, one with Dupuytren's contracture, and two with high-voltage electrical injuries).Thirty-six pedicled limb fillet flaps were used in 35 cases. In 12 cases, salvage of above-knee or below-knee amputated stumps was achieved with a plantar neurovascular island pedicled flap. In seven other cases, sacral, pelvic, groin, hip, abdominal wall, or lumbar defects were reconstructed with fillet-of-thigh or entire-limb fillet flaps. In five cases, defects of shoulder, head, neck, and thoracic wall were covered with upper-arm fillet flaps. In nine cases, defects of the forefoot were covered by adjacent dorsal or plantar fillet flaps. In two other cases, defects of the upper arm or the proximal forearm were reconstructed with a forearm fillet. The average size of these defects was 512 cm2. Thirteen major joints were salvaged, three stumps were lengthened, and nine foot or forefoot amputations were prevented. One partial flap necrosis occurred in a patient with a fillet-of-sole flap. In another case, wound infection required revision and above-knee amputation with removal of the flap.Nine free plantar fillet flaps were performed-five for coverage of amputation stumps and four for sacral pressure sores. Seven free forearm fillet flaps, one free flap of forearm and hand, and one forearm and distal upper-arm fillet flap were performed for defect coverage of the shoulder and neck area. The average size of these defects was 432 cm2. Four knee joints were salvaged and one above-knee stump was lengthened. No flap necrosis was observed. One patient died of acute respiratory distress syndrome 6 days after surgery.Major complications were predominantly encountered in small finger and toe fillet flaps. Overall complication rate, including wound dehiscence and secondary grafting, was 18 percent. This complication rate seems acceptable. Major complications such as flap loss, flap revision, or severe infection occurred in only 7.5 percent of cases. The majority of our cases resulted from severe trauma with infected and necrotic soft tissues, disseminated tumor disease, or ulcers in elderly, multimorbid patients.On the basis of these data, a classification was developed that facilitates multicenter comparison of procedures and their clinical success. Fillet flaps facilitate reconstruction in difficult and complex cases. The spare part concept should be integrated into each trauma algorithm to avoid additional donor-site morbidity and facilitate stump-length preservation or limb salvage.     

Sequence requirements in the catalytic core of the "10-23" DNA enzyme

A systematic mutagenesis study of the "10-23" DNA enzyme was performed to analyze the sequence requirements of its catalytic domain. Therefore, each of the 15 core nucleotides was substituted separately by the remaining three naturally occurring nucleotides. Changes at the borders of the catalytic domain led to a dramatic loss of enzymatic activity, whereas several nucleotides in between could be exchanged without severe effects. Thymidine at position 8 had the lowest degree of conservation and its substitution by any of the other three nucleotides caused only a minor loss of activity. In addition to the standard nucleotides (adenosine, guanosine, thymidine, or cytidine) modified nucleotides were used to gain further information about the role of individual functional groups. Again, thymidine at position 8 as well as some other nucleotides could be substituted by inosine without severe effects on the catalytic activity. For two positions, additional experiments with 2-aminopurine and deoxypurine, respectively, were performed to obtain information about the specific role of functional groups. In addition to sequence-function relationships of the DNA enzyme, this study provides information about suitable sites to introduce modified nucleotides for further functional studies or for internal stabilization of the DNA enzyme against endonucleolytic attack.     

Interactions of Pex7p and Pex18p/Pex21p with the peroxisomal docking machinery: implications for the first steps in PTS2 protein import

Peroxisomal PTS2-dependent matrix protein import starts with the recognition of the PTS2 targeting signal by the import receptor Pex7p. Subsequently, the formed Pex7p/cargo complex is transported from the cytosol to the peroxisomal docking complex, consisting of Pex13p and Pex14p. In Saccharomyces cerevisiae, the latter event is thought to require the redundant Pex18p and Pex21p. Here we mapped the Pex7p interaction domain of Pex13p to its N-terminal 100 amino acids. Pex18p and Pex21p also interacted with this region, albeit only in the presence of Pex7p. Expression of an N-terminally deleted version of Pex13p in a pex13delta mutant failed to restore growth on fatty acids due to a specific defect in the import of PTS2-containing proteins. We further show by yeast two-hybrid analysis, coimmunoprecipitation, and in vitro binding assays that Pex7p can bind Pex13p and Pex14p in the absence of Pex18p/Pex21p. The PTS2 protein thiolase was shown to interact with Pex14p but not with Pex13p in a Pex7p- and Pex18p/Pex21p-dependent manner, suggesting that only Pex14p binds cargo-loaded PTS2 receptor. We also found that the cytosolic Pex7p/thiolase-containing complex includes Pex18p. This complex accumulated in docking mutants but was absent in cells lacking Pex18p/Pex21p, indicating that Pex18p/Pex21p are required already before the docking event.     

Design of antisense oligonucleotides stabilized by locked nucleic acids

The design of antisense oligonucleotides containing locked nucleic acids (LNA) was optimized and compared to intensively studied DNA oligonucleotides, phosphorothioates and 2′-O-methyl gapmers. In contradiction to the literature, a stretch of seven or eight DNA monomers in the center of a chimeric DNA/LNA oligonucleotide is necessary for full activation of RNase H to cleave the target RNA. For 2′-O-methyl gapmers a stretch of six DNA monomers is sufficient to recruit RNase H. Compared to the 18mer DNA the oligonucleotides containing LNA have an increased melting temperature of 1.5–4°C per LNA depending on the positions of the modified residues. 2′-O-methyl nucleotides increase the T_m by only <1°C per modification and the T_m of the phosphorothioate is reduced. The efficiency of an oligonucleotide in supporting RNase H cleavage correlates with its affinity for the target RNA, i.e. LNA > 2′-O-methyl > DNA > phosphorothioate. Three LNAs at each end of the oligonucleotide are sufficient to stabilize the oligonucleotide in human serum 10-fold compared to an unmodified oligodeoxynucleotide (from t_1/2 = ~1.5 h to t_1/2 = ~15 h). These chimeric LNA/DNA oligonucleotides are more stable than isosequential phosphorothioates and 2′-O-methyl gapmers, which have half-lives of 10 and 12 h, respectively.