Manufacturing DNA microarrays from unpurified PCR products

For the production of DNA microarrays from PCR products, purification of the the DNA fragments prior to spotting is a major expense in cost and time. Also, a considerable amount of material is lost during this process and contamination might occur. Here, a protocol is presented that permits the manufacture of microarrays from unpurified PCR products on aminated surfaces such as glass slides coated with the widely used poly(L-lysine) or aminosilane. The presence of primer molecules in the PCR sample does not increase the non-specific signal upon hybridisation. Overall, signal intensity on arrays made of unpurified PCR products is 94% of the intensity obtained with the respective purified molecules. This slight loss in signal, however, is offset by a reduced variation in the amount of DNA present at the individual spot positions across an array, apart from the considerable savings in time and cost. In addition, a larger number of arrays can be made from one batch of amplification products.     

Enhanced efficiency through nuclear localization signal fusion on phage PhiC31-integrase: activity comparison with Cre and FLPe recombinase in mammalian cells

The integrase of the phage ΦC31 recombines an attP site in the phage genome with a chromosomal attB site of its Streptomyces host. We have utilized the integrase-mediated reaction to achieve episomal and genomic deletion of a reporter gene in mammalian cells, and provide the first comparison of its efficiency with other recombinases in a new assay system. This assay demonstrated that the efficiency of ΦC31-integrase is significantly enhanced by the C-terminal, but not the N-terminal, addition of a nuclear localization signal and becomes comparable with that of the widely used Cre/loxP system. Furthermore, we found that the improved FLP recombinase, FLPe, exhibits only 10% recombination activity on chromosomal targets as compared with Cre, whereas the Anabaena derived XisA recombinase is essentially inactive in mammalian cells. These results provide the first demonstration that a nuclear localisation signal and its position within a recombinase can be important for its efficiency in mammalian cells and establish the improved ΦC31-integrase as a new tool for genome engineering.     

Substrate-based design of reversible Pin1 inhibitors

Human Pin1, a peptidyl-prolyl cis/trans isomerase with high specificity to -Ser/Thr(PO(3)H(2))-Pro- motifs, is required for cell cycle progression. In an effort to design reversible Pin1 inhibitors by using a substrate structure based approach, a panel of peptides were applied to systematically analyze the minimal structural requirements for Pin1 substrate recognition. Pin1 catalysis (k(cat)/K(m) < 5 mM(-1) s(-1)) for Ala-Pro, Ser-Pro, and Ser(PO(3)H(2))-Pro was detected using direct UV-visible spectrophotometric detection of prolyl isomerization, while weak competitive inhibition of Pin1 by these dipeptides was observed (K(i) > 1 mM). Substrates with chain lengths extending from either the P2 to P1' or the P1 to P2' subsite gave k(cat)/K(m) values of 100 mM(-1) s(-1) for Ala-Ser(PO(3)H(2))-Pro and 38 mM(-1) s(-1) for Ser(PO(3)H(2))-Pro-Arg. For both Pin1 and its yeast homologue Ess1, the optimal subsite recognition elements comprise five amino acid residues with the essential Ser(PO(3)H(2)) in the middle position. The resulting substrate Ac-Ala-Ala-Ser(PO(3)H(2))-Pro-Arg-NH-4-nitroanilide possesses a very low cis/trans interconversion barrier in the presence of either Pin1 or Ess1, with k(cat)/K(m) = 9300 mM(-1) s(-1) and 12000 mM(-1) s(-1), respectively. The D-Ser(PO(3)H(2)) residue preceding proline could serve as a substrate-deactivating determinant without compromising ground state affinity. Similarly, substitution of the amide bond preceding proline with a thioxo amide bond produces a potent inhibitor. Pin1 is reversibly inhibited by such substrate analogue inhibitors with IC(50) values in the low micromolar range. The D-amino acid containing inhibitor also exhibits remarkable stability against phosphatase activity in cell lysate.     

Control of receptor-induced signaling complex formation by the kinetics of ligand/receptor interaction

Tumor necrosis factor (TNF) exists both as a membrane-integrated type II precursor protein and a soluble cytokine that have different bioactivities on TNFR2 (CD120b) but not on TNFR1 (CD120a). To identify the molecular basis of this disparity, we have investigated receptor chimeras comprising the cytoplasmic part of Fas (CD95) and the extracellular domains of the two TNF receptors. The membrane form of TNF, but not its soluble form, was capable of inducing apoptosis as well as activation of c-Jun N-terminal kinase and NF-kappaB via the TNFR2-derived chimera. In contrast, the TNFR1-Fas chimera displayed strong responsiveness to both TNF forms. This pattern of responsiveness is identical to that of wild type TNF receptors, demonstrating that the underlying mechanisms are independent of the particular type of the intracellular signaling machinery and rather are controlled upstream of the intracellular domain. We further demonstrate that the signaling strength induced by a given ligand/receptor interaction is regulated at the level of adaptor protein recruitment, as shown for FADD, caspase-8, and TRAF2. Since both incidents, strong signaling and robust adapter protein recruitment, are paralleled by a high stability of individual ligand-receptor complexes, we propose that half-lives of individual ligand-receptor complexes control signaling at the level of adaptor protein recruitment.     

cGMP-mediated signaling via cGKIalpha is required for the guidance and connectivity of sensory axons

Previous in vitro studies using cGMP or cAMP revealed a cross-talk between signaling mechanisms activated by axonal guidance receptors. However, the molecular elements modulated by cyclic nucleotides in growth cones are not well understood. cGMP is a second messenger with several distinct targets including cGMP-dependent protein kinase I (cGKI). Our studies indicated that the alpha isoform of cGKI is predominantly expressed by sensory axons during developmental stages, whereas most spinal cord neurons are negative for cGKI. Analysis of the trajectories of axons within the spinal cord showed a longitudinal guidance defect of sensory axons within the developing dorsal root entry zone in the absence of cGKI. Consequently, in cGKI-deficient mice, fewer axons grow within the dorsal funiculus of the spinal cord, and lamina-specific innervation, especially by nociceptive sensory neurons, is strongly reduced as deduced from anti-trkA staining. These axon guidance defects in cGKI-deficient mice lead to a substantial impairment in nociceptive flexion reflexes, shown using electrophysiology. In vitro studies revealed that activation of cGKI in embryonic dorsal root ganglia counteracts semaphorin 3A-induced growth cone collapse. Our studies therefore reveal that cGMP signaling is important for axonal growth in vivo and in vitro.     

Resorbable plate osteosynthesis of dislocated or pathological mandibular fractures: a prospective clinical trial of two amorphous L-/DL-lactide copolymer 2-mm miniplate systems

The purpose of this study was to evaluate the indication for resorbable miniplates in traumatic and pathological mandibular fractures. Two resorbable miniplate systems, the 2.0-mm MacroSorb (Macropore, San Diego, Calif.) and the PolyMax (Synthes, Oberdorf, Switzerland), were prospectively used in 24 mandibular osteosyntheses. Made from amorphous 70:30 poly-L/DL-lactide, amorphous copolymer plates have not yet been evaluated for mandibular osteosyntheses. The main advantage of the amorphous copolymer-structure is continuous hydrolysis through water penetration into the implant body during the first 6 months. Hydrolysis breaks the copolymer chains into smaller particles that later become degraded through phagocytotic cells. Twelve patients, aged 13 to 83 years, were treated after providing informed consent. Fourteen dentate patients with moderately dislocated traumatic fractures and two edentulous atrophic and dislocated traumatic mandibular fractures were treated. Two patients with pathological fractures due to osteomyelitis received osteosynthesis after sequestrectomy. Histological specimens of the plates, screws, and surrounding soft tissues were taken after 3, 6, 9, and 12 months in secondary dental implant operations. A total of 22 osteosyntheses healed well without clinical or radiological signs of dislocation, insufficient or excess formation of callus, pseudarthrosis, or plate fracture. The follow-up time ranged from 4 to 22 months. One patient with osteomyelitis worsened because of widespread osseous infection, and one with atrophic fracture developed a bland fibrous pseudarthrosis. The histological specimens showed a moderate inflammatory foreign body reaction. No sinuous drainage or clinically apparent inflammation occurred. The presented osteosynthesis systems showed reliable stability for mandibular osteosynthesis in cooperative patients; however, two treatment failures occurred (8 percent). Disadvantages of the resorbable osteosyntheses were costs, greater diameter, screw breakage, and the need to place the screws vertically to the plate. The use of resorbable osteosyntheses in dislocated fractures should be further evaluated in controlled studies.     

Activation of the DNA-dependent protein kinase by drug-induced and radiation-induced DNA strand breaks

The DNA-dependent protein kinase (DNA-PK) is a DNA-end activated protein kinase that is required for efficient repair of DNA double-strand breaks (DSBs) and for normal resistance to ionizing radiation. DNA-PK is composed of a DNA-binding subunit, Ku, and a catalytic subunit, DNA-PKcs (PRKDC). We have previously shown that PRKDC is activated when the enzyme interacts with the terminal nucleotides of a DSB. These nucleotides are often damaged when DSBs are introduced by anticancer agents and could therefore prevent recognition by DNA-PK. To determine whether DNA-PK could recognize DNA strand breaks generated by agents used in the treatment of cancer, we damaged plasmid DNA with anticancer drugs and ionizing radiation. The DNA breaks were tested for the ability to activate purified DNA-PK. The data indicate that DSBs produced by bleomycin, calicheamicin and two types of ionizing radiation ((137)Cs gamma rays and N(7+) ions: high and low linear energy transfer, respectively) activate DNA-PK to levels matching the kinase activation obtained with simple restriction endonuclease-induced DSBs. In contrast, the protein-linked DSBs produced by etoposide and topoisomerase II failed to bind and activate DNA-PK. Our findings indicate that DNA-PK recognizes DSBs regardless of chemical complexity but cannot recognize the protein-linked DSBs produced by etoposide and topoisomerase II.     

Development of a Listeria monocytogenes EGDe partial proteome reference map and comparison with the protein profiles of food isolates

A partially annotated proteome reference map of the food pathogen Listeria monocytogenes was developed for exponentially growing cells under standardized, optimal conditions by using the sequenced strain EGDe (serotype 1/2a) as a model organism. The map was developed by using a reproducible total protein extraction and two-dimensional (2-D) polyacrylamide gel electrophoresis analysis procedure, and it contained 33 identified proteins representing the four main protein functional classes. In order to facilitate analysis of membrane proteins, a protein compartmentalization procedure was assessed. The method used provided partial fractionation of membrane and cytosolic proteins. The total protein 2-D profiles of three serotype 1/2a strains and one serotype 1/2b strain isolated from food were compared to the L. monocytogenes EGDe proteome. An average of 13% of the major protein spots in the food strain proteomes were not matched in the strain EGDe proteome. The variation was greater for the less intense spots, and on average 28% of these spots were not matched. Two of the proteins identified in L. monocytogenes EGDe were missing in one or more of the food isolates. These two proteins were proteins involved in the main glycolytic pathway and in metabolism of coenzymes and prosthetic groups. The two corresponding genes were found by PCR amplification to be present in the four food isolates. Our results show that the L. monocytogenes EGDe reference map is a valuable starting point for analyses of strains having various origins and could be useful for analyzing the proteomes of different isolates of this pathogen.