Release of granule proteins from eosinophils cultured with IL-5.

Eosinophils isolated from normal individuals were cultured in the presence of human rIL-5 (hrIL-5) for up to 14 days, and the effects of this exposure were determined. First, the hrIL-5-cultured eosinophils were activated and degranulated more readily than freshly isolated eosinophils. For example, eosinophils cultured for 7 days with hrIL-5 released 30 and 10% of granule eosinophil-derived neurotoxin (EDN) when exposed to Sepharose 4B beads coupled to secretory IgA and IgG, respectively, whereas freshly isolated eosinophils released only 19 and 4%, respectively, of their EDN in response to the same stimuli. Degranulation of hrIL-5-cultured eosinophils was not augmented by further exposure to hrIL-5, whereas degranulation of freshly isolated cells to secretory IgA and IgG beads was increased by exposure to hrIL-5. Second, eosinophils cultured with hrIL-5 had prolonged viability in vitro. For example, after four days of culture with 50 U/ml of hrIL-5, 86% of eosinophils were viable compared to 12% in medium alone. Third, hrIL-5-cultured eosinophils became hypodense, and electron microscopy showed that they contained granules with core and matrix lucency and with evidence of granule fusion. Fourth, hrIL-5-cultured eosinophils spontaneously lost 30 to 60% of their EDN, eosinophil cationic protein, and eosinophil peroxidase and about 50% of their eosinophil granule major basic protein content compared to freshly isolated eosinophils, and all four of the granule proteins were released into the culture medium. Fifth, detailed studies of eosinophils cultured in hrIL-5 showed that 89 +/- 10% of the starting quantity of EDN could be recovered at 7 days. Whereas 99 +/- 1% of the EDN at day 0 was cell associated, by 7 days 60 +/- 9% was in the cell supernatants. Thus, hrIL-5 activates eosinophils, increases their viability, decreases their density, and their content of granule proteins and causes release of the granule proteins into culture fluids. The striking loss of granule proteins during culture with hrIL-5 may be an important mechanism for deposition of these cationic toxins in various diseases where IL-5 plays a role.     

Evidence for a direct interaction of Rev protein with nuclear envelop mRNA-translocation system.

The interaction of the Rev protein from human immunodeficiency virus type 1 (HIV-1) with the nucleocytoplasmic mRNA-transport system was investigated. In gel-shift assay, the recombinant Rev protein used in this study selectively bound to the Rev-responsive element (RRE) region of HIV-1 env-specific RNA. Nitrocellulose-filter-binding studies and Northern/Western-blotting experiments revealed an association constant of approximately 1 x 10(10) M-1. The Rev protein also strongly bound to isolated nuclear envelopes from H9 cells, containing the poly(A)-binding site (= mRNA carrier) and the nucleoside triphosphatase (= NTPase), which are thought to be involved in nuclear export of poly(A)-rich mRNA. Binding of 125I-Rev to a 110-kDa nuclear-envelope protein, the putative mRNA carrier, could be demonstrated in in vitro experiments. Both efflux of cellular poly(A)-rich RNA, such as actin RNA [but not efflux of poly(A)-free RNA] from isolated nuclei and the nuclear-envelope NTPase activity were strongly inhibited by Rev protein. On the other hand, transport of viral env RNA, containing the Rev-responsive element, was increased in the presence of Rev. Studying the release of RNA from closed nuclear-envelope vesicles containing entrapped RNA, the action of Rev was found to occur at the level of translocation of RNA through the nuclear pore. Evidence is presented that Rev down-regulates the NTPase-driven transport of mRNA lacking the RRE, most likely via binding to the mRNA carrier within the envelope. In contrast to the efflux of RRE-free RNA, ATP-dependent efflux of RRE-containing RNA from resealed nuclear-envelope vesicles was found to be increased, if the RNA was entrapped in the vesicles together with Rev protein. In addition, it was found that phosphorylated Rev, which is transported together with RRE-containing RNA out of the vesicles, becomes dephosphorylated during transport. In the vesicle experiments it is demonstrated for the first time that a protein selectively channels a specific mRNA across the nuclear-envelope pore complex.     

S-neurons and not L-neurons are the source of GABAergic action in the ocellar retina

Summary Electrophysiological evidence obtained with current- and voltage clamp experiments from single L-neurons of the ocellar nerve of locust (Locusta migratoria) questions a direct synaptic feedback from these neurons onto the photoreceptors. The synaptic currents recorded under voltage clamp reflected the photoresponse of the L-neuron, despite the fact it developed no synaptic activity under these conditions. This result is contrary to GABAergic feedback models proposed in the literature. Electrophysiological recordings, as well as immunocytochemistry revealing GABA and glutamate decarboxylase, indicated a possible contribution of S-neurons in such a feedback system. A population of probable S-neurons whose somas were in the pars intercerebralis adjacent to the ocellar nerve tracts was heavely labelled. About 10 fibres entered each tract and formed a dense network of fine arborizations within the ocellar plexiform layer. L-neurons showed no GABA-immunoreactivity. Based on these data a new model for GABAergic feedback is proposed and discussed.     

CYCLOHEXIMIDE AS A MEDIA AMENDMENT FOR ENUMERATING BACTERIAL POPULATIONS IN ANIMAL FEEDS

The present study was designed to evaluate cycloheximide as a potential media amendment for differential bacterial and fungal enumeration of animal feeds. The objectives of this study were to determine the effect of cycloheximide on bacterial growth rates and to evaluate its efficacy for the reduction of indigenous spreading fungi when enumerating bacterial populations in three types of feeds and after short or long-term storage. Escherichia coli, Bacillus cereus, Staphylococcus aureus, Listeria monocytogenes and Pseudomonas fluorescens were grown in tryptic soy broth containing cycloheximide to determine its effect on bacterial specific growth rates. Growth rates of B. cereus and S. aureus were significantly decreased by the addition of 600 and 1000 mg/L cycloheximide respectively, but other pure cultures were not significantly influenced by cycloheximide addition. Intrinsic bacterial populations from feed were not significantly affected by cycloheximide additions at concentrations from 10 to 300 mg/L, but the indigenous spreading molds from feeds were significantly decreased by these cycloheximide concentrations and were decreased below detection levels by 300 mg/L of cycloheximide. The addition of 300 mg/L of cycloheximide effectively eliminates fungal growth for accurate enumeration of bacterial populations in feeds.     

INDIGENOUS POULTRY FEED MICROFLORA RESPONSE TO ETHYL ALCOHOL AND BUFFERED PROPIONIC ACID ADDITION

The present study was designed to compare ethyl alcohol with buffered propionic acid feed treatment on the survival of indigenous poultry feed bacteria and fungi. The aerobic bacterial poultry feed populations were not substantially reduced by either ethyl alcohol or buffered propionic acid treatments. Likewise, indigenous poultry feed fungal populations also were not markedly reduced by buffered propionic acid treatment of the feed but fungal poultry feed populations exposed to ethyl alcohol treatments were significantly lower (P<0.05) than fungal populations recovered from either control and buffered propionic acid treated feeds. Ethyl alcohol treatment may have potential for reducing fungal contamination in poultry feed.     

High endogenous activated protein C levels attenuates bleomycin‐induced pulmonary fibrosis

Coagulation activation accompanied by reduced anticoagulant activity is a key characteristic of patients with idiopathic pulmonary fibrosis (IPF). Although the importance of coagulation activation in IPF is well studied, the potential relevance of endogenous anticoagulant activity in IPF progression remains elusive. We assess the importance of the endogenous anticoagulant protein C pathway on disease progression during bleomycin‐induced pulmonary fibrosis. Wild‐type mice and mice with high endogenous activated protein C APC levels (APC^high) were subjected to bleomycin‐induced pulmonary fibrosis. Fibrosis was assesses by hydroxyproline and histochemical analysis. Macrophage recruitment was assessed immunohistochemically. In vitro, macrophage migration was analysed by transwell migration assays. Fourteen days after bleomycin instillation, APC^high mice developed pulmonary fibrosis to a similar degree as wild‐type mice. Interestingly, Aschcroft scores as well as lung hydroxyproline levels were significantly lower in APC^high mice than in wild‐type mice on day 28. The reduction in fibrosis in APC^high mice was accompanied by reduced macrophage numbers in their lungs and subsequent in vitro experiments showed that APC inhibits thrombin‐dependent macrophage migration. Our data suggest that high endogenous APC levels inhibit the progression of bleomycin‐induced pulmonary fibrosis and that APC modifies pulmonary fibrosis by limiting thrombin‐dependent macrophage recruitment.     

Production and characterization of polyclonal and monoclonal antibodies against Aflatoxin B1 oxime-BSA in an enzyme-linked immunosorbent assay

From a single aflatoxin B₁ oxime — bovine serum albumin conjugate, polyclonal and monoclonal antibody preparations were produced. The four rabbit polyclonal antisera were specific for aflatoxin Bi in a microtitration plate enzyme — linked immunosorbent assay. The monoclonal antibodies showed a wide range of differing specificities, recognizing, for example, aflatoxins B₁, B₂, G₁ and G₂; B₁ and B₂; B₁ and G₁; and G₁ alone. No antibody preparations reacted with aflatoxin M₁. The significance of these results to the strategy of anti-aflatoxin antibody production for use in quantitative enzyme immunoassays is discussed.