Effect of inhibition of thromboxane production on the leukotriene D4-mediated bronchoconstriction in the guinea pig

Leukotriene D₄ (LTD₄) administered intravenously to anesthetized, spontaneously breathing guinea pigs elicited decreases in dynamic lung compliance (C_dyn) and airway conductance (G_AW) with a maximal response achieved at 0.5 min. Simultaneously, plasma levels of thromboxane metabolite, TxB₂, and the prostacyclin metabolite, 6-keto-PGF_1α, increased 10-fold over pre-LTD₄ levels. Pretreatment of the guinea pigs with meclofenamic acid delayed the onset of the LTD₄-induced bronchoconstriction, antagonized the magnitude of the decreases in C_dyn and G_AW, and blocked the increase in plasma TxB₂ and 6-keto-PGF_1α levels. The thromboxane synthetase inhibitor, UK 37,248, suppressed the LTD₄-induced bronchoconstriction, while it completely blocked TxB₂ production without significantly affecting 6-keto-PGF_1α. The SRS-A end organ antagonist, FPL 55712, blocked both the LTD₄-induced bronchoconstriction and the production of the arachidonic acid metabolites. These results suggest that thromboxane A₂ plays an important role in mediating part of the bronchoconstriction elicited by intravenously administered LTD₄ in the guinea pig.     

Experimental autoallergic sialadenitis in the LEW rat. I. Parameters of disease induction

Experimental autoallergic sialadenitis (EAS) is an autoimmune mononuclear cell infiltration of the submandibular salivary gland that results in tissue destruction and glandular dysfunction. A previous report has described an animal model of induced EAS in LEW rats following sensitization with allogeneic WF submandibular gland (SMG). The present study extends this observation to an EAS disease model induced following sensitization of LEW rats with syngeneic LEW SMG. Furthermore, we describe the characterization of the mononuclear cells in the glandular infiltrates, evaluate the production of autoantibodies, and establish the parameters important for reproducible induction of EAS. Our results demonstrate that EAS can be induced in a completely syngeneic system and the histopathology of disease induction in the syngeneic and allogeneic model systems is similar. Helper/inducer (CD4+) and suppressor/cytotoxic (CD8+) T-cell subsets are the dominant cell types in the salivary mononuclear cell infiltrate. An anti-duct autoantibody was found in the serum of virtually all LEW rats with EAS. Although closely associated with disease development, the presence of this antibody was not a prerequisite for development of histopathologic disease. Induction of disease in both the syngeneic and allogeneic models of EAS is dependent upon administration of Bordetella pertussis at the time of sensitization. Finally, the histopathology of the cellular infiltrates in both the allogeneic and syngeneic models of EAS resemble those observed in the salivary tissues of Sj?gren's patients. While there are several differences between EAS in the LEW rat and the full expression of Sj?gren's syndrome, EAS may serve as a model to study the salivary gland component of this complex human disease.     

[3H]MK-801 Binding to N-Methyl-d-Aspartate Receptors Solubilized from Rat Brain: Effects of Glycine Site Ligands, Polyamines, Ifenprodil, and Desipramine

The N-methyl-D-aspartate (NMDA) receptor is thought to contain several distinct binding sites that can regulate channel opening. In the present experiments, the effects of ligands for these sites have been examined on [3H]MK-801 binding to a soluble receptor preparation, which had been passed down a gel filtration column to reduce the levels of endogenous small-molecular-weight substances. Glycine site agonists, partial agonists, and antagonists gave effects similar to those observed in membranes [EC50 values (in microM): glycine, 0.31; D-serine, 0.20; D-cycloserine, 1.46; (+)-HA-966, 4.06; and 7-chlorokynurenic acid, 1.81]. Spermine and spermidine enhanced [3H]MK-801 binding to the soluble receptor preparation (EC50, 4.3 and 20.1 microM, respectively), whereas putrescine and cadaverine gave small degrees of inhibitions. When spermine and spermidine were tested under conditions where [3H]MK-801 binding approached equilibrium, their ability to enhance [3H]MK-801 binding was much reduced, a result suggesting that the polyamines increase the rate to equilibrium. Putrescine antagonised the effects of spermine. Ifenprodil reduced [3H]MK-801 binding under both equilibrium and nonequilibrium conditions, although the high-affinity component of inhibition described in membranes was not observed. Ifenprodil antagonised spermine effects in an apparently noncompetitive manner. Desipramine was able to give total inhibition of specific [3H]MK-801 binding under nonequilibrium conditions with an IC50 of 4 microM, and this value was unaltered when [3H]MK-801 binding was allowed to reach equilibrium. These results suggest that the sites mediating the effects of glycine and its analogues, polyamines and desipramine are integral components of the NMDA receptor protein.     

Chromosomal integration of plasmid DNA by homologous recombination in Enterococcus faecalis and Lactococcus lactis subsp. lactis hosts harboring Tn919.

Integration of pCI192, a pBR322-derived vector plasmid containing homology to the chromosomally located conjugative transposon Tn919 was observed in two strains that harbor Tn919, namely, Enterococcus faecalis GF590 and Lactococcus lactis subsp. lactis CH919. Hybridization analysis indicated that single-copy integration of the plasmid had occurred at low frequency. The Tn919::plasmid structure was conjugated from an E. faecalis donor to a L. lactis recipient, although at lower frequencies than was Tn919. Segregation of the tetracycline and chloramphenicol resistance markers during conjugation was observed. The integration strategy described allows for DNA manipulations to be performed in an easily manipulated model host strain with the subsequent transfer of integrated structures by conjugation to any strain capable of receiving Tn919. The results indicate that homologous recombination events may be used to introduce plasmid-encoded genes to the lactococcal chromosome.     

Physicochemical properties of cloned nucleocapsid protein from HIV. Interactions with metal ions

The nucleocapsid (NC) protein (p15) of the human immunodeficiency virus (HIV) has been cloned and overproduced (under the control of a phage T7 promoter) in soluble form in an Escherichia coli host. The soluble NC protein is a fusion protein containing 15 amino acids from the T7 gene 10 and 7 amino acids from the HIV p24 protein at the N-terminus to make a protein of 171 amino acids. The plasmid containing the fusion gene is designated p15DF. A homogeneous product has been isolated from the induced cells and, when isolated under aerobic conditions, contains 0.3-0.5 mol of Zn/mol of protein and has only 2 titratable SH groups. Reduction and refolding in the presence of Zn(II) yields a protein containing 2.0 mol of Zn/mol of protein and 6 titratable SH groups. On the other hand, if the cells are sonicated in 2 mM CdCl2 and purified at pH 5.0, an unoxidized protein containing 2 mol of Cd/mol of protein is obtained. The Cd(II) ions can be exchanged with Zn(II), Co(II), or 113Cd(II). The Co(II)2 NC protein shows d-d electronic transitions at 695 nm [epsilon = 675 M-1 cm-1 per Co(II)] and 640 nm [epsilon = 825 M-1 cm-1 per Co(II)] compatible with regular tetrahedral geometry around both Co(II) ions. The Co(II)2 and Cd(II)2 NC proteins show intense charge-transfer bands in the near-UV, at 355 nm (epsilon = approximately 4000 M-1 cm-1) and 310 nm (epsilon = approximately 8000 M-1 cm-1) for the Co(II) protein and 255 nm (epsilon = approximately 10(4) M-1 cm-1) for the Cd(II)2 NC protein, compatible with -S- coordination. 113Cd NMR of the 113Cd(II)2 NC protein shows two 113Cd NMR signals at 659 and 640 ppm, respectively, each integrating to approximately 1 Cd(II) ion. The downfield chemical shifts suggest coordination of each 113Cd(II) ion to 3 sulfur donor atoms. The spectroscopic data fully support the prediction that the NC protein binds metal ions to each of the tandem repeats of the -Cys-X2-Cys-X4-His-X4-Cys- sequence contained in the N-terminal half of the molecule. 113Cd NMR shows, however, that the sites are not identical. Isolation of the NC protein under standard aerobic conditions results in oxidation of the sulfhydryl groups and loss of the coordinated Zn(II) ions, while preparation of the NC protein as the Cd(II) derivative at low pH protects the sulfhydryl groups from oxidation.     

Follicle-stimulating hormone pulse amplitude decreases with the onset of the breeding season in the mare

The relationship between daily mean FSH concentrations in serum and the pattern of FSH detected by frequent sampling for 12-h periods (samples every 15 min) was examined in five mares during the transition into the breeding season. The five mature anestrous mares were exposed to a natural increase in daylength. Blood samples were collected daily from February 1 until the first ovulation of the breeding season (April 14 +/- 3.7 days, Mean +/- SEM). Periods of frequent blood collection were performed every two weeks. Blood samples were obtained daily by jugular venipuncture or jugular cannula (frequent samples). Mean daily concentrations of FSH in serum determined by RIA decreased during seasonal transition. Patterns of FSH in serum detected by frequent sampling were pulsatile. FSH pulse amplitude decreased during seasonal transition, and the decrease in amplitude was associated with the decrease in mean serum FSH concentrations. This decrease in FSH pulse amplitude may reflect an involvement of a follicular product from developing follicles or a change in hypothalamic stimulation of pituitary FSH release.     

Leaf shelter-building caterpillars harness forces generated by axial retraction of stretched and wetted silk

Leaf shelter-building caterpillars generate most of the force required to pull leaf surfaces together by stretching silk strands while spinning. Axially retractive forces produced by columns of stretched strands enabled caterpillars in our study to generate forces as great as 0.3 Newtons (i.e., a 30-g force). We found that caterpillar silk also contracts instantly when wetted, producing an additional, though smaller, axially retractive force. Contraction ratios (final length/ original length) of the wetted silk of 19 species ranged from 0.21 to 0.93 and were smallest among species that use their silk to make leaf shelters. Our study, the first to identify the specific sources of the energy harnessed by caterpillars to tie, roll, or fold leaves, indicates that silk properties and caterpillar behavior have coevolved to facilitate the leaf shelter-building process.