Co-precipitation molecules hemopexin and transferrin may be key molecules for fibrillogenesis in TTR V30M amyloidogenesis

The disease model of familial amyloidotic polyneuropathy—7.2-hMet30 mice—manifests amyloid deposition that consists of a human amyloidogenic mutant transthyretin (TTR) (TTR V30M). Our previous study found amyloid deposits in 14 of 27 7.2-hMet30 mice at 21–24 months of age. In addition, non-fibrillar TTR deposits were found in amyloid-negative 7.2hMet30 mice. These results suggested that TTR amyloidogenesis required not only mutant TTR but also an additional factor (or factors) as an etiologic molecule. To determine the differences in serum proteome in amyloid-positive and amyloid-negative mice in the 7.2-hMet30 model, we used proteomic analyses and studied serum samples obtained from these mice. Hemopexin (HPX) and transferrin (Tf) were detected in the serum samples from amyloid-positive mice and were also found in amyloid deposits via immunohistochemistry, but serum samples from amyloid-negative mice did not contain HPX and Tf. These two proteins were also not detected in non-fibrillar TTR deposits. In addition, in silico analyses suggested that HPX and Tf facilitate destabilization of TTR secondary structures and misfolding of TTR. These results suggest that HPX and Tf may be associated with TTR amyloidogenesis after fibrillogenesis in vivo.     

Preparation of monoclonal antibodies against planarian organs and the effect of fixatives

Monoclonal antibodies were obtained against all part of the body of the planarian Dugesia japonica japonica except tthe nerve cord, though parts of the enteric canal were also non reactive. The effect of a variety of fixatives (acetone, Bouin, formalin) used on planarian tissues prior to screening with antibodies, was assessed.     

Decreased levels of alpha 1(I) procollagen mRNA in dermal fibroblasts grown on fibrin gels and in response to fibrinopeptide B

We have investigated human neonatal fibroblast synthetic activity in response to fibrin substrates and components of fibrin formation and degradation. Greater than threefold downregulation of procollagen mRNA levels was seen 24 hours after fibroblasts were grown on fibrin gels as compared to tissue culture plastic. This downregulation occurred in both reptilase-generated fibrin (retention of fibrinopeptide B) and thrombin-generated fibrin (loss of both fibrinopeptide A and B). However, fibroblasts grown on fibrin retained their capacity to respond to the stimulatory action of transforming growth factor (TGF)-beta 1. Fibroblasts seeded on reptilase-generated fibrin displayed an abnormal morphology manifested by dendritic appearance and cell rounding, while fibroblast attachment was enhanced by 30% on thrombin-generated fibrin substrate (P < 0.02). Fibrinopeptides A and B, which are generated during fibrin formation, increased and decreased procollagen mRNA levels, respectively. Tissue plasminogen activator (t-PA) increased procollagen mRNA and TGF-beta 1 levels as early as 6 hours after cells were grown on tissue culture plastic, but this stimulation did not occur in cells cultured on a fibrin substrate. We conclude that alpha 1(I) procollagen mRNA levels in cultures of human dermal fibroblasts are consistently down-regulated by a fibrin substrate and are directly and profoundly influenced by complex interactions between components involved in the formation and removal of fibrin. © 1995 Wiley-Liss, Inc.     

Activation of phospholipase D by prostaglandin F2α in rat luteal cells and effects of inhibitors of arachidonic acid metabolism

In rat luteal cells labeled with (³H]oleic acid, PGF_2α-stimulated phospholipase D (PLD) activation was investigated. The PLD activity was detected by measuring the accumulation of [³H]phosphatidylethanol (PtdEt) in the presence of ethanol. PGF_2α stimulated PtdEt accumulation at concentrations of more than 100 nM in the presence of ethanol. However, PtdEt accumulation did not change in the absence of ethanol. PGF_2α (1 μM) increased PtdEt accumulation after 1 min, and the accumulation reached a plateau by 2–3 min. These results indicate that PGF_2α activates PLD in rat luteal cells. U-73122, a phospholipase C (PLC) inhibitor, and staurosporine, a protein kinase C (PKC) inhibitor, did not inhibit PGF_2α-stimulated [³H]PtdEt accumulation. These results suggest that PGF_2α-induced PLD activation is different from PLC-PKC systems. We reported previously that PGF_2α stimulated the release of arachidonic acid. The effects of indomethacin, nordihydroguaiaretic acid (NDGA), and 5,8,11,14-eicosatetraynoic acid (ETYA), inhibitors of arachidonic acid metabolism, on PGF_2α-stimulated PtdEt accumulation were examined. Pretreatment with indomethacin enhanced PGF_2α-induced PtdEt accumulation. In contrast, pretreatment with NDGA and ETYA inhibited PGF_2α-induced PtdEt accumulation. It is suggested that PGF_2α-stimulated PLD activation is mediated via lipoxygenase products.     

Enhanced Expression of Fc Receptors on Neutrophils from Calves with Leukocyte Adhesion Deficiency

The expression of Fc receptors for immunoglobulin G(IgG) and concanavalin A (con A)-binding receptors, luminol-dependent chemiluminescent (LDCL) responses, and the effect of anti-bovine IgG on LDCL responses were evaluated in neutrophils from Holstein calves with leukocyte adhesion deficiency (BLAD). Neutrophils from affected calves showed a 2.1- to 2.5-fold increase in Fc receptor expression compared with those of control calves by flow cytometric analysis. Con A-binding activities of neutrophils from affected calves were similar to those of control calves. Neutrophils from a calf with BLAD, when stimulated with zymosan opsonized with bovine serum (OPZ), heat-aggregated bovine IgG (Agg-bovine IgG), sheep red blood cells (SRBC) sensitized with anti-SRBC antibody (SRBC-anti-SRBC Ab), or con A had LDCL responses of 36 (P<0.05), 77, 126 and 119% of peak LDCL values of controls, respectively. The NBT-reducing value of neutrophils from a calf with BLAD when stimulated with Agg-bovine IgG after pretreatment with anti-bovine IgG was 116.5% of the values of neutrophils from control calves, but the difference was not significant. The LDCL responses of neutrophils from a control calf and a calf with BLAD stimulated with OPZ were inhibited markedly by pre-incubation with anti-bovine IgG antiserum at concentrations ranging from 1.25 to 20 or 40 μg/ml. Although an increase in Fc receptor expression on neutrophils from calves with BLAD was observed, the LDCL responses stimulated with SRBC-anti-SRBC Ab and NBT-reducing activity stimulated with Agg-bovine IgG after pretreatment with anti-bovine IgG did not correlate significantly with the increased Fc receptor expression. These results support that neutrophil functions mediated by the Fc receptors are associated synergistically with the presence of the complement receptor type 3 (CR3)(CD11b/CD18).     

Probiotic effect of Lactobacillus sp. DS-12 in flounder (Paralichthys olivaceus)

Pea aphids were reared aseptically in the laboratory and fed on strains of Erwinia aphidicola, Erwinia herbicola, Klebsiella pneumoniae, Escherichia coli and bacterium W, an aphid enterobacterium, by mixing with a synthetic diet to determine whether these bacteria have pathogenicity to the insect. It turned out that Er. aphidicola and Er. herbicola grew in aphid gut, while K. pneumoniae, Es. coli and bacterium W were effectively eliminated. In the case of Er. aphidicola, ingestion of a single dose of 10(3) CFU/ml was enough to infect aphid gut and to prevent post-final ecdysis growth of the insect. As a result, the body weight of the infected aphids was significantly reduced (p<0.01). A concentration of 10(5) CFU/ml of Er. aphidicola caused aphid mortality. In contrast, Er. herbicola demanded 10(5) CFU/ml to colonize in aphid gut, and seemed to have no effect on insect health.     

Cytokinin/Auxin Control of Apical Dominance in Ipomoea nil

Although the concept of apical dominance control by the ratioof cytokinin to auxin is not new, recent experimentation withtransgenic plants has given this concept renewed attention.In the present study, it has been demonstrated that cytokinintreatments can partially reverse the inhibitory effect of auxinon lateral bud outgrowth in intact shoots of Ipomoea nil. Althoughless conclusive, this also appeared to occur in buds of isolatednodes. Auxin inhibited lateral bud outgrowth when applied eitherto the top of the stump of the decapitated shoot or directlyto the bud itself. However, the fact that cytokinin promotiveeffects on bud outgrowth are known to occur when cytokinin isapplied directly to the bud suggests different transport tissuesand/or sites of action for the two hormones. Cytokinin antagonistswere shown in some experiments to have a synergistic effectwith benzyladenine on the promotion of bud outgrowth. If theratio of cytokinin to auxin does control apical dominance, thenthe next critical question is how do these hormones interactin this correlative process? The hypothesis that shoot-derivedauxin inhibits lateral bud outgrowth indirectly by depletingcytokinin content in the shoots via inhibition of its productionin the roots was not supported in the present study which demonstratedthat the repressibility of lateral bud outgrowth by auxin treatmentsat various positions on the shoot was not correlated with proximityto the roots but rather with proximity to the buds. Resultsalso suggested that auxin in subtending mature leaves as wellas that in the shoot apex and adjacent small leaves may contributeto the apical dominance of a shoot. (Received September 24, 1996; Accepted March 16, 1997)     

Decrease of bone mineral density and muscle and/or strength in the leg during 20 days horizontal bed rest

Ten individuals underwent 20 days of horizontal bed rest for this study of the influence of muscle mass and strength on bone mineral density. Muscle mass volume and cross sectional area were measured using magnetic resonance imaging and bone mineral density was determined by dual energy X-ray absorptiometry before and after bed rest. Measurements were made at various parts of the leg, including the knee. Gender differences were also determined. Results are presented and discussed.     

Effect of leukotriene C4 exposure on ciliated cells of the nasal mucosa

To clarify the effects of leukotriene C4 (LTC4) on human ciliated epithelium, ciliary activity of the ethmoid sinus mucosa was measured photoelectrically in tissue culture. At concentrations ranging from 10⁻⁶M to 10⁻⁹M, LTC4 showed minimal effects on the ciliated epithelium during the initial 30 minutes of exposure; thereafter, ciliary inhibition was observed in a concentration- and time-dependent manner. Irrigation of the mucosa with culture medium 15 minutes after exposure prevented the LTC4-induced ciliary inhibition. However, irrigation 60 minutes after exposure failed to inhibit 10⁻⁸M LTC4-induced ciliary dysfunction and mucosal damage. The LTC4-induced ciliary inhibition was blocked in the presence of FPL-55712 and/or Ly-171883, both leukotriene receptor antagonists. L-serine and sodium tetraborate complex (SBC), a γ-glutamyl transpeptidase (γ-GTP) inhibitor, also inhibited the LTC4-induced ciliary inhibition. These findings indicate that LTC4 is converted to LTD4 by γ-GTP during 60 minutes of exposure, and LTC4 itself has minimal direct effects on the ciliated cells.