The dynamics of pAL2-1 homologous linear plasmids in Podospora anserina

We have investigated the properties of the newly synthesized proton-pump inhibitor, 3-butyryl-8-methoxy-4-[(2-thiophenyl)amino]quinoline (YJA20379-6), on gastric mucosal proton-pump (H+/K+-ATPase) activity, gastric acid secretion and gastroduodenal lesions in experimental rats. YJA20379-6 markedly inhibited H+/K+-ATPase activity in rabbit isolated gastric mucosal microsomes, confirming its classification as a proton-pump inhibitor. The inhibitory efficacy of YJA20379-6 on the proton pump was approximately 14-times higher than that of omeprazole at pH 7.4. YJA20379-6 given intraduodenally had a potent inhibitory effect on gastric secretion in pylorus-ligated rats (ED50 22.9 mg kg(-1)) but was less active than omeprazole. Pretreatment of rats with YJA20379-6 dose-dependently protected the gastric mucosa from damage induced by water-immersion stress, indomethacin and absolute ethanol, and the duodenal mucosa from damage induced by mepirizole. Repeated administration of YJA20379-6 also dose-dependently accelerated the spontaneous healing of acetic acid-induced gastric ulcers. These results suggest that YJA20379-6 has potent anti-secretory and anti-ulcer effects which are exerted by suppression of H+/K+-ATPase activity in gastric parietal cells. YJA20379-6 might be useful for the clinical treatment of peptic ulcer diseases.     

A computer-controlled spraying-freezing apparatus for millisecond time-resolution electron cryomicroscopy

The application of gene therapy techniques to the clinical problem of coronary restenosis has generated tremendous attention and enthusiasm. Use of gene transfer technology to prevent a common intractable illness would represent a watershed event for human gene therapy. However, the time is not yet right to initiate gene therapy trials for restenosis. The biology of restenosis is incompletely understood, catheter-based gene delivery is poorly adapted to the coronary circulation, and current gene transfer vectors are ill-suited for safe and effective gene delivery to the coronary artery wall. Basic research designed to overcome these obstacles is currently more appropriate than the initiation of clinical trials.     

The nonstructural small glycoprotein sGP of Ebola virus is secreted as an antiparallel-orientated homodimer

An earlier report indicated that a 26-amino-acid peptide (SA), comprised of the nuclear localization signal (NLS) of fibroblast growth factor-1 (FGF-1) and a membrane-permeable peptide, was able to stimulate DNA synthesis after it was taken up by NIH3T3 fibroblasts. Here, we report that SA, but not a mutant with the NLS motif destroyed, induced DNA synthesis in BALB/c3T3 murine fibroblasts, human vascular endothelial (HUVE) cells, and primary cultured hepatocytes, although the activity was weaker than that of FGF-1. The kinetics of SA-induced DNA synthesis and G1 cyclin expression were similar to those elicited by FGF-1, indicating that SA induces cell cycle progression. Kinetic analysis also suggested that SA stimulates only a fraction of the DNA replication in BALB/c3T3 cells. At high cell densities, SA-induced G1 cyclin expression and DNA synthesis were more strongly inhibited than those induced by FGF-1. SA did not induce cell division in HUVE and BALB/c3T3 cells and did not interfere with FGF-1-stimulated proliferation of HUVE cells. These results indicate that SA is able to partially induce cell cycle progression through a contact-inhibition sensitive signaling pathway, but it is insufficient to support cell mitosis. We also suggest that signaling by SA does not interfere with that of FGF-1.     

Engineering the S1' subsite of trypsin: design of a protease which cleaves between dibasic residues

The serine protease trypsin was converted into a site-specific protease which hydrolyzes peptides between dibasic residues. Trypsin exhibits a high S1 specificity for Arg and Lys residues. However, the S1' specificity of trypsin is very broad, with only a slight preference for hydrophobic residues in P1'. We replaced Lys60 with Glu and Asp to introduce a high specificity for basic residues into the S1' site of trypsin. Both mutations cause a dramatic increase in the S1' specificity for Arg and Lys as measured by acyl transfer reactions. In K60E, the preference for Arg increases 70-fold while the preference for P1'-Lys increases 12-fold. In contrast, the preferences for other P1' residues either decrease slightly or remain the same. Thus, K60E prefers P1'-Arg over most other P1' residues by 2 orders of magnitude. Similar results are obtained when P1' specificity is measured in peptide cleavage assays. K60D exhibits an S1' specificity profile very similar to that of K60E, although the P1'-Arg preference is reduced by a factor of 2.5. Molecular modeling studies suggest that the high S1' specificity for Arg in K60E may be due to the formation of a salt bridge between Glu60 and the P1'-Arg of the substrate.     

The specificity of prolyl endopeptidase from Flavobacterium meningoseptum: mapping the S' subsites by positional scanning via acyl transfer

The S1'-S3' subsite specificity of prolyl endopeptidase from Flavobacterium meningoseptum was studied by acyl transfer to libraries of amino acid amides and peptides. Whereas the S1' and S3' subsites influence the specificity for the amino component by approximately one order of magnitude, the S2' subsite possesses a markedly higher specificity. Besides the high specificity for hydrophobic residues at P1'-P3', proline was efficiently bound by the S2' and S3' subsites of the enzyme. In contrast, no binding of P1' proline-containing peptides was observed. It could be demonstrated that the specificity of the S' subsite is not restricted to L-amino acids. Effective P'-S' interactions were also found for beta- and gamma-amino acids indicating that the enzyme does not form close contacts to the backbone of P1' and P2' amino acid residues.     

The Wiskott-Aldrich syndrome

In 1937, Wiskott described three brothers with congenital thrombocytopenia, bloody diarrhea, eczema, and recurrent ear infections. Seventeen years later, Aldrich showed X-linked (a gene carried on the X chromosome) inheritance. Subsequently, the characteristic immune defects of Wiskott-Aldrich syndrome (WAS) were reported, including lymphopenia, lymphocyte depletion in the thymus, T-dependent pericortical areas of lymph nodes, defective delayed type hypersensitivity, and failure to produce antibodies to polysaccharides and to a variety of bacterial, protein, and viral antigens. The consistent platelet abnormalities were explained by ineffective thrombocytopoiesis. The increased risk of autoimmune diseases and malignancies was recognized. In addition to the classic WAS phenotype, a milder form designated as hereditary X-linked thrombocytopenia (XLT) has been described. The genes for both WAS and XLT have been mapped to Xp11.22 and sequence analysis has identified mutations of the same gene in both phenotypes. The gene coding for the WAS protein (WASP) is composed of 12 exons containing 1,823 base pairs and encodes a 502-amino acid protein. WASP is expressed in the cytoplasm of all hematopoietic stem cell-derived lineages. Although the precise function of WASP is unknown, several unique binding domains have been identified, and WASP appears to play a critical role in signal transduction by interacting with SH3-containing molecules and in the regulation of the cytoskeletal reorganization. The identification of the WASP gene allows the diagnosis of WAS on a molecular basis, carrier detection, and prenatal diagnosis. Treatment is largely symptomatic and includes antibiotics, prophylactic intravenous immunoglobulin (i.v.IG) and splenectomy in selected cases to reduce hemorrhages. Stem cell transplantation corrects the defect and should be considered in younger patients.     

Cellular uptake and localization of liposomal-methylphosphonate oligodeoxynucleotides

Nuclease digestion and intracellular delivery are major factors limiting the potential use of oligodeoxynucleotides as antisense molecules. Structural analogues of phosphodiester oligodeoxynucleotides, such as phosphorothioates and methylphosphonates, are resistant to nuclease degradation and can still bind to their mRNA targets. However, their limited ability to escape from the endosomal/lysosomal compartments and reach the intracellular sites of action have dampened their potential clinical application. To circumvent this problem we have incorporated methylphosphonate oligodeoxynucleotides into liposomes. We found that the level of uptake of liposome-incorporated methylphosphonate oligodeoxynucleotides is time and concentration dependent. Maximal up take occurred at 8 h when 4-8 microM liposome-incorporated methylphosphonate oligodeoxynucleotides was added. Approximately 50% of liposome-incorporated methylphosphonate oligodeoxynucleotides were retained in cells after 24 h of incubation. Using fluorescent microscopy, intracellular fluorescence could be seen within 2.5 h of incubation. Diffused fluorescence was found throughout the cytoplasm, suggesting that the liposome-incorporated methylphosphonate oligodeoxynucleotides were not confined within the endosomal/lysosomal structures. We conclude that liposomes can effectively deliver methylphosphonate oligodeoxynucleotides to the cytoplasm, which is the major intracellular site of action for translational arrest.     

Transcutaneous lower blepharoplasty

Transcutaneous lower blepharoplasty as a standard procedure for cosmetic lower blepharoplasty is being replaced by transconjunctival blepharoplasty and laser resurfacing techniques. Nevertheless, there remain specific indications for the transcutaneous approach and its modifications. The accomplished facial plastic surgeon will be able to choose the appropriate technique based on the patient's concerns, the eyelid pathology, and his or her clinical experience.