Role of plasminogen in propagation of scrapie

To investigate whether plasminogen may feature in scrapie infection, we inoculated plasminogen-deficient (Plg(-/-)), heterozygous plasminogen-deficient (Plg(+/-)), and wild-type (Plg(+/+)) mice by the intracerebral or intraperitoneal (i.p.) route with the RML scrapie strain and monitored the onset of neurological signs of disease, survival time, brain, and accumulation of scrapie disease-associated forms of the prion protein (PrP(Sc)). Only after i.p. inoculation, a slight, although significant, difference in survival (P < 0.05) between Plg(-/-) and Plg(+/+) mice was observed. Neuropathological examination and Western blot analysis were carried out when the first signs of disease appeared in Plg(+/+) animals (175 days after i.p. inoculation) and when mice reached the terminal stage of illness. At the onset of symptoms, PrP(Sc) accumulation was higher in the brain and spleen of Plg(+/+) and Plg(+/-) mice than in those of Plg(-/-) mice, and these differences were paralleled by differences in the severity of spongiform changes and astrogliosis in the cerebral cortex and subcortical gray structures. Immunohistochemical analysis of the spleens before inoculation did not show any impairment of the immune system affecting follicular dendritic or lymphoid cells in Plg(-/-) mice. Once the disease progressed and mice began to die of infection, differences were no longer apparent in either brains or spleens. In conclusion, our data indicate that plasminogen has no major effect on the survival of scrapie agent-infected mice.     

Oxygen-dependent PAF receptor binding and intracellular signaling in ovine fetal pulmonary vascular smooth muscle

Circulating levels of platelet-activating factor (PAF) are high in the fetus, and PAF is active in maintaining high PVR in fetal hypoxia (Ibe BO, Hibler S, Raj J. J Appl Physiol 85: 1079-1085, 1998). PAF synthesis by fetal pulmonary vascular smooth muscle cells (PVSMC) is high in hypoxia, but how oxygen tension affects PAF receptor (PAF-r) binding in PVSMC is not known. We studied the effect of oxygen tension on PAF-r binding and signaling in fetal PVSMC. PAF binding was saturable. PAF-r density (B(max): fmol/10(6) cells; means +/- SE, n = 6), 25.2 +/- 0.77 during hypoxia (Po(2) <40 Torr), was higher than 13.9 +/- 0.44 during normoxia (Po(2) approximately 100 Torr). K(d) was twofold lower in hypoxia than normoxia. PAF-r protein expression, 35-40% greater in hypoxia, was inhibited by cycloheximide, a protein synthesis inhibitor, suggesting translational regulation. IP(3) release, an index of PAF-r-mediated cell signaling, was greater in hypoxia (EC(50): hypoxia, 2.94 +/- 0.61; normoxia, 5.85 +/- 0.51 nM). Exogenous PAF induced 50-90% greater intracellular calcium flux in cells during hypoxia, indicating hypoxia augments PAF-r-mediated cell signaling. PAF-r phosphorylation, with or without 5 nM PAF, was 40% greater in hypoxia. These data show 1) hypoxia upregulates PAF-r binding, PAF-r phosphorylation, and PAF-r-mediated intracellular signaling, evidenced by augmented IP(3) production and intracellular Ca(2+) flux; and 2) hypoxia-induced PAF-r phosphorylation results in activation of PAF-r-mediated signal transduction. The data suggest the fetal hypoxic environment facilitates PAF-r binding and signaling, thereby promoting PAF-mediated pulmonary vasoconstriction and maintenance of high PVR in utero.     

Evaluation of dog semen quality after slow (biological freezer) or rapid (nitrogen vapours) freezing

Three ejaculates were collected from each of five dogs. After initial evaluation, the sperm-rich fractions were diluted to 100 x 10(6) spermatozoa x mL(-1) in two steps with an egg yolk-TRIS extender containing a final concentration of 5% glycerol and 0.5% Equex STM paste. Half of the 0.5 mL straws obtained from each ejaculate were frozen on nitrogen vapours (4 cm above the liquid surface) ("rapid freezing"), while the other half was frozen in a biological freezer at a rate of 0.5 degrees C x min(-1) between 5 degrees C and -10 degrees C and of 8 degrees C x min(-1) between -10 degrees C and -60 degrees C, followed by immersion in liquid nitrogen ("slow freezing"). After an average storage of 30 days, the straws were thawed in a water-bath at 37 degrees C for 1 min. Progressive motility was subjectively estimated hourly for 8 h on semen incubated at 38 degrees C. Immediately after thawing and after 2 h of incubation, motility parameters were also measured by a motility analyser. Sperm membrane function and chromatin stability were assessed immediately post-thaw, using the hypo-osmotic swelling test and acridine orange staining, respectively. Slow freezing significantly improved total post-thaw motility, which showed a slower decline over time, although spermatozoal average path and straight line velocity were lower compared to the fast rate. Also the number of intact membrane spermatozoa was significantly higher in slow-frozen samples while the proportion of spermatozoa with single-stranded DNA was minimal after both freezing procedures.     

The power and potential of doxorubicin-DNA adducts

Doxorubicin (trade name Adriamycin) is a widely used anticancer agent which exhibits good activity against a wide range of tumors. Although the major mode of action appears to be normally as a topoisomerase II poison, it also exhibits a number of other cellular responses, one of which is the ability to form adducts with DNA. For adduct formation doxorubicin must react with cellular formaldehyde to form an activated Schiff base which is then able to form an aminal (N-C-N) linkage to the exocyclic amino group of guanine residues. The mono-adducts form primarily at G of 5'-GCN-3' sequences where the chromophore of the drug is intercalated between the C and N base pair. The structure of the adducts has have been well defined by 2D NMR, mass spectrometry and X-ray crystallography. The formation of these anthracycline adducts in cells grown in culture has been unequivocally demonstrated. The source of formaldehyde in cells can be endogenous, provided by coadministration of prodrugs that release formaldehyde or by prior complexation of anthracyclines with formaldehyde. Since the adducts appear to be more cytotoxic than doxorubicin alone, and also less susceptible to drug-efflux forms of resistance, they offer new approaches to improving the anticancer activity of the anthracyclines.     

Haemophilus somnus possesses two systems for acquisition of transferrin-bound iron

Haemophilus somnus strain 649 was found to acquire iron from ovine, bovine, and goat transferrins (Tfs). Expression of Tf receptors, as evaluated by solid-phase binding assays, required the organisms to be grown under iron-restricted conditions in the presence of Tf. Competition binding assays revealed the presence of two distinct Tf-binding receptor systems, one specific for bovine Tf and the other capable of binding all three ruminant Tfs. Affinity isolation procedures using total membranes yielded three putative bovine Tf-binding polypeptides and one putative ovine and goat Tf-binding polypeptide. PCR amplification followed by DNA sequence analyses revealed that H. somnus strain 649 possesses genes that encode a bipartite TbpA-TbpB receptor along with a homolog of the Histophilus ovis single-component TbpA receptor. Expression of TbpB and the single-component TbpA would appear to be subject to a form of phase variation involving homopolymeric nucleotide tracts within the structural genes.     

A Simple Geometrical Pattern for the Branching Distribution of the Bronchial Tree, Useful to Estimate Optimality Departures

The design of the bronchial tree has largely been proposed as a model of optimal design from a physical-functional perspective. However, the distributive function of the airway may be more related to a geometrical than a physical problem. The bronchial tree must distribute a three dimensional volume of inspired air on a two dimensional alveolar surface, included in a limited volume. It is thus valid to ask whether an optimal bronchial tree from a physical perspective is also optimum from a geometrical point of view. In this paper we generate a simple geometric model for the branching pattern of the bronchial tree, deducing relationships that permit estimation of the departures from the geometrical optimum of each bifurcation. We also, for comparative purposes, estimate the departures from the physical optimum. From the geometrical assumptions: i) a symmetrical dichotomic fractal design, ii) with minimum volume and iii) maximum dispersion of the terminal points; and several simulations we suggest that the optimality is characterized by a bifurcation angle theta approximately 60 degrees and a length reduction scale gamma = (1/2)(1/3) = 0.7937. We propose distances from the physical and geometrical optimality defined as Euclidean distances from the expected optima. We show how the advanced relationships and the distances can be used to estimate departures from the optimality in bronchographs of four species. We found lower physical and geometrical departures in the distal zone than those of the proximal zones, as well as lower physical than geometrical departures from optimality.     

Table of Contents of Volume 52 - 2004

Table of Contents

Table of Contents of Volume 52 - 2004     

Analysis of autoreactive CD4 T cells in experimental autoimmune encephalomyelitis after primary and secondary challenge using MHC class II tetramers

Experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, is primarily mediated by CD4 T cells specific for Ags in the CNS. Using MHC class II tetramers, we assessed expansion and phenotypic differentiation of polyclonal self-reactive CD4 T cells during EAE after primary and secondary challenge with the specific Ag. After EAE induction in SJL mice with proteolipid protein 139-151, CNS-specific T cells up-regulated activation markers and expanded in the draining lymph nodes and in the spleen. Less than 20% of total autoreactive T cells entered the CNS simultaneously with Th cells of other specificities. Almost all tetramer-positive cells in the CNS were activated and phenotypically distinct from the large peripheral pool. When EAE was induced in Ag-experienced mice, disease symptoms developed earlier and persisted longer; autoreactive T cells were more rapidly activated and invaded the CNS earlier. In striking contrast to specific CTLs that respond after secondary viral challenge, the absolute numbers of autoreactive CD4 T cells were not increased, indicating that the accelerated autoreactivity in Ag-experienced mice is not related to higher frequencies of autoreactive CD4 T cells.