The Effect of Cauda Equina Constriction on Nitric Oxide Synthase Activity

Nitric oxide synthase (NOS) activity was studied in the gray and white matter regions of the spinal cord 2 and 5 days after multiple cauda equina constrictions of the central processes of L7-Co5 dorsal root ganglia neurons. The results show considerable differences in enzyme activity in the thoracic, upper lumbar, lower lumbar, and sacral segments. Increased NOS activity was observed at 2 days after multiple cauda equina constrictions in the dorsal, lateral, and ventral columns of the lower lumbar segments and in the ventral column of the upper lumbar segments. The values returned to control levels within 5 postconstriction days. In the lateral columns of thoracic segments taken 2 and 5 days after surgery, NOS activity was enhanced by 54% and 55% and in the upper lumbar segments by 130% and 163%, respectively. Multiple cauda equina constrictions performed surgically for 2 and 5 days caused a significant increase in NOS activity predominantly in the gray matter regions of thoracic segments. A quite different response was found 5 days postconstriction in the upper lumbar segments, where the enzyme activity was significantly decreased in the dorsal horn, intermediate zone, and ventral horn. No such extreme differences could be seen in the lower lumbar segments, where NOS activity was significantly enhanced only in the ventral horn. The data correspond with a higher number of NOS immunoreactive somata, quantitatively evaluated in the ventral horn of the lower lumbar segments at 5 days after multiple cauda equina constrictions. While the great region-dependent heterogeneity in NOS activity seen 2 and 5 days after multiple cauda equina constrictions is quite apparent and suggestive of an active role played by nitric oxide in neuroprotective or neurotoxic processes occurring in the gray and white matter of the spinal cord, the extent of damage or the degree of neuroprotection caused by nitric oxide in compartmentalized gray and white matter in this experimental paradigm would be possible only using longer postconstriction periods.     

Changes in glycosidase activities during galactoglucomannan oligosaccharide inhibition of auxin induced growth

The inhibition of 2,4-D-induced elongation growth by galactoglucomannan oligosaccharides (GGMOs) in pea stem segments (Pisum sativum L. cv. Tyrkys) after 18 h of incubation results in changes of extracellular, intracellular and cell wall glycosidase activities (beta-D-glucosidase, beta-D-mannosidase, beta-D-galactosidase, beta-D-xylosidase, alpha-D-galactosidase, and alpha-L-arabinosidase). GGMOs lowered the glycosidase activities in the extracellular fraction, while in the cell wall fractions their activities were markedly increased. The intracellular enzyme alpha-d-galactosidase increased while the beta-d-galactosidase decreased in activity in response to the GGMO treatment. Extracellular enzymes showed low values of activities in comparison with intracellular and cell wall glycosidases. It is evident that GGMOs can alter auxin induced elongation and glycosidase activities in different compartments of the cell, however, the mode and site of their action remains unclear.     

Veterinary parasitic vaccines: pitfalls and future directions

Most available antiparasitic drugs are safe, cheap and highly effective against a broad spectrum of parasites. However, the alarming increase in the number of parasite species that are resistant to these drugs, the issue of residues in the food chain and the lack of new drugs stimulate development of alternative control methods in which vaccines would have a central role. Parasite vaccines are still rare, but there are encouraging signs that their number will increase in the next decade. The modern paradigm is that an understanding of parasite genes will lead to the identification of useful antigens, which can then be produced in recombinant systems developed as a result of the huge investment in biotechnology. However, we should also continue to devote efforts to basic research on the host-parasite interface.     

Involvement of sialoadhesin in entry of porcine reproductive and respiratory syndrome virus into porcine alveolar macrophages

Porcine reproductive and respiratory syndrome virus (PRRSV) shows a very restricted tropism for cells of the monocyte/macrophage lineage. It enters cells via receptor-mediated endocytosis. A monoclonal antibody (MAb) that is able to block PRRSV infection of porcine alveolar macrophages (PAM) and that recognizes a 210-kDa protein (p210) was described previously (MAb41D3) (X. Duan, H. Nauwynck, H. Favoreel, and M. Pensaert, J. Virol. 72:4520-4523, 1998). In the present study, the p210 protein was purified from PAM by immunoaffinity using MAb41D3 and was subjected to internal peptide sequencing after tryptic digestion. Amino acid sequence identities ranging from 56 to 91% with mouse sialoadhesin, a macrophage-restricted receptor, were obtained with four p210 peptides. Using these peptide data, the full p210 cDNA sequence (5,193 bp) was subsequently determined. It shared 69 and 78% amino acid identity, respectively, with mouse and human sialoadhesins. Swine (PK-15) cells resistant to viral entry were transfected with the cloned p210 cDNA and inoculated with European or American PRRSV strains. Internalized virus particles were detected only in PK-15 cells expressing the recombinant sialoadhesin, demonstrating that this glycoprotein mediated uptake of both types of strains. However, nucleocapsid disintegration, like that observed in infected Marc-145 cells as a result of virus uncoating after fusion of the virus with the endocytic vesicle membrane, was not observed, suggesting a block in the fusion process. The ability of porcine sialoadhesin to mediate endocytosis was demonstrated by specific internalization of MAb41D3 into PAM. Altogether, these results show that sialoadhesin is involved in the entry process of PRRSV in PAM.     

The structure of a cold-adapted family 8 xylanase at 1.3 A resolution. Structural adaptations to cold and investgation of the active site

Enzymes from psychrophilic organisms differ from their mesophilic counterparts in having a lower thermostability and a higher specific activity at low and moderate temperatures. The current consensus is that they have an increased flexibility, enhancing accommodation and transformation of the substrates at low energy costs. Here we describe the structure of the xylanase from the Antarctic bacterium Pseudoalteromonas haloplanktis at 1.3 A resolution. Xylanases are usually grouped into glycosyl hydrolase families 10 and 11, but this enzyme belongs to family 8. The fold differs from that of other known xylanases and can be described as an (alpha/alpha)(6) barrel. Various parameters that may explain the cold-adapted properties were examined and indicated that the protein has a reduced number of salt bridges and an increased exposure of hydrophobic residues. The crystal structures of a complex with xylobiose and of mutant D144N were obtained at 1.2 and 1.5 A resolution, respectively. Analysis of the various substrate binding sites shows that the +3 and -3 subsites are rearranged as compared to those of a family 8 homolog, while the xylobiose complex suggests the existence of a +4 subsite. A decreased acidity of the substrate binding cleft and an increased flexibility of aromatic residues lining the subsites may enhance the rate at which substrate is bound.     

HIV-1 integrase forms stable tetramers and associates with LEDGF/p75 protein in human cells

We studied human immunodeficiency virus, type 1 (HIV-1) integrase (IN) complexes derived from nuclei of human cells stably expressing the viral protein from a synthetic gene. We show that in the nuclear extracts IN exists as part of a large distinct complex with an apparent Stokes radius of 61 A, which dissociates upon dilution yielding a core molecule of 41 A. We isolated the IN complexes from cells expressing FLAG-tagged IN and demonstrated that the 41 A core is a tetramer of IN, whereas 61 A molecules are composed of IN tetramers associated with a cellular protein with an apparent molecular mass of 76 kDa. This novel integrase interacting protein was found to be identical to lens epithelium-derived growth factor (LEDGF/p75), a protein implicated in regulation of gene expression and cellular stress response. HIV-1 IN and LEDGF co-localized in the nuclei of human cells stably expressing IN. Furthermore, recombinant LEDGF robustly enhanced strand transfer activity of HIV-1 IN in vitro. Our findings indicate that the minimal IN molecule in human cells is a homotetramer, suggesting that at least an octamer of IN is required to accomplish coordinated integration of both retroviral long terminal repeats and that LEDGF is a cellular factor involved in this process.     

Free radical scavengers can differentially modulate the genotoxicity of amsacrine in normal and cancer cells

Amsacrine is an acridine derivative drug applied in haematological malignancies. It targets topoisomerase II enhancing the formation of a cleavable DNA-enzyme complex and leading to DNA fragmentation in dividing cancer cells. Little is known about other modes of the interaction of amsacrine with DNA, by which it could affect also normal cells. Using the alkaline comet assay, we showed that amsacrine at concentrations from the range 0.01 to 10 microM induced DNA damage in normal human lymphocytes, human promyelocytic leukemia HL-60 cells lacking the p53 gene and murine pro-B lymphoid cells BaF3 expressing BCR/ABL oncogene measured as the increase in percentage tail DNA. The effect was dose-dependent. Treated cells were able to recover within a 120-min incubation. Amifostine at 14 mM decreased the level of DNA damage in normal lymphocytes, had no effect on the HL-60 cells and potentiated the DNA-damaging effect of the drug in BCR/ABL-transformed cells. Vitamin C at 10 and 50 microM diminished the extent of DNA damage in normal lymphocytes, but had no effect in cancer cells. Pre-treatment of the cells with the nitrone spin trap, N-tert-butyl-alpha-phenylnitrone or ebselen, which mimics glutathione peroxidase, reduced the extent of DNA damage evoked by amsacrine in all types of cells. The cells exposed to amsacrine and treated with endonuclease III and 3-methyladenine-DNA glycosylase II, the enzymes recognizing oxidized and alkylated bases, respectively, displayed greater extent of DNA damage than those not treated with these enzymes. The results obtained suggest that free radicals may be involved in the formation of DNA lesions induced by amsacrine. The drug can also methylate DNA bases. Our results indicate that the induction of secondary malignancies should be taken into account as diverse side effects of amsacrine. Amifostine may potentate DNA-damage effect of amsacrine in cancer cells and decrease this effect in normal cells and Vitamin C can be considered as a protective agent against DNA damage in normal cells.     

Effects of metallic silver island films on resonance energy transfer between N,N'-(dipropyl)-tetramethyl- indocarbocyanine (Cy3)- and N,N'-(dipropyl)-tetramethyl- indodicarbocyanine (Cy5)-labeled DNA

Resonance energy transfer (RET) is typically limited to distances below 60 A, which can be too short for some biomedical assays. We examined a new method for increasing the RET distances by placing donor- and acceptor-labeled DNA oligomers between two slides coated with metallic silver particles. A N,N'-(dipropyl)-tetramethylindocarbocyanine donor and a N,N'-(dipropyl)-tetramethylindodicarbocyanine acceptor were covalently bound to opposite 5' ends of complementary 23 base pair DNA oligomers. The transfer efficiency was 25% in the absence of silver particles or if only one slide was silvered, and it increased to an average value near 64% between two silvered slides. The average value of the Forster distance increased from 58 to 77 A. The energy transfer data were analyzed with a model assuming two populations of donor-acceptor pairs: unaffected and affected by silver island films. In an affected fraction of about 28%, the apparent energy transfer efficiency is near 87% and the Forster distance increases to 119 A. These results suggest the use of metallic silver particles to increase the distances over which RET occurs in biomedical and biotechnology assays.     

Identification and characterization of B"-subunits of protein phosphatase 2 A in Xenopus laevis oocytes and adult tissues.

Protein phosphatase 2A is a phosphoserine/threonine phosphatase implicated in many cellular processes. The core enzyme comprises a catalytic and a PR65/A-subunit. The substrate specificity and subcellular localization are determined by a third regulatory B-subunit (PR55/B, PR61/B' and PR72/130/B"). To identify the proteins of the B" family in Xenopus laevis oocytes, a prophase Xenopus oocyte cDNA library was screened using human PR130 cDNA as a probe. Three different classes of cDNAs were isolated. One class is very similar to human PR130 and is probably the Xenopus orthologue of PR130 (XPR130). A second class of clones (XN73) is identical to the N-terminal part of XPR130 but ends a few amino acids downstream of the putative splicing site of PR130. To investigate how this occurs, the genomic structure of the human PR130 gene was determined. This novel protein does not act as a PP2A subunit but might compete with the function of PR130. The third set of clones (XPR70) is very similar to human PR48 but has an N-terminal extension. Further analysis of the human EST-database and the human PR48 gene structure, revealed that the human PR48 clone published is incomplete. The Xenopus orthologue of PR48 encodes a protein of 70 kDa which like the XPR130, interacts with the A-subunit in GST pull-down assays. XPR70 is ubiquitously expressed in adult tissues and oocytes whereas expression of XPR130 is very low in brain and oocytes. Expression of XN73 mainly parallels XPR130 with the exception of the brain.     

Characterization of serracin P,a phage-tail-like bacteriocin,and its activity against Erwinia amylovora,the fire blight pathogen

Serratia plymithicum J7 culture supernatant displayed activity against many pathogenic strains of Erwinia amylovora, the causal agent of the most serious bacterial disease of apple and pear trees, fire blight, and against Klebsiella pneumoniae, Serratia liquefaciens, Serratia marcescens, and Pseudomonas fluorescens. This activity increased significantly upon induction with mitomycin C. A phage-tail-like bacteriocin, named serracin P, was purified from an induced culture supernatant of S. plymithicum J7. It was found to be the only compound involved in the antibacterial activity against sensitive strains. The N-terminal amino acid sequence analysis of the two major subunits (23 and 43 kDa) of serracin P revealed high homology with the Fels-2 prophage of Salmonella enterica, the coliphages P2 and 168, the phiCTX prophage of Pseudomonas aeruginosa, and a prophage of Yersinia pestis. This strongly suggests a common ancestry for serracin P and these bacteriophages.