Survival of <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> in Wilted and Additive-Treated Grass Silage

Grass was field-dried to 3 different dry matter (DM) levels (200, 430 and 540 g/kg) and inoculated with 10⁶–10⁷ cfu/g of a Listeria monocytogenes strain sharing a phagovar occasionally involved in food-borne outbreaks of listeriosis. Formic acid (3 ml/kg) or lactic acid bacteria (8·10⁵/g) with cellulolytic enzymes were applied only to forages with low and intermediate DM levels. Forages were ensiled in laboratory silos (1700 ml) and were stored at 25°C for 30 or 90 days. After 90 days of storage, L. monocytogenes could not be detected in any silo, except one with the high dry matter grass without additive. After 30 days of storage, between 10² and 10⁶ cfu L. monocytogenes/g silage were isolated from the untreated silages. Increasing the DM content from 200 to 540 g/kg did not reduce listeria counts possibly because of the lower production of fermentation acids (higher pH). In silages treated with additives, counts of L. monocytogenes were always lower than in silages without additive. In wet silages (DM 200 g/kg) both additives were effective, but in the wilted silages (DM 430 g/kg) only the bacterial additive reduced listeria counts below detection level. Listeria counts were highly correlated to silage pH (r = 0.92), the concentration of lactic acid (r = -0.80) and the pooled amount of undissociated acids (r = -0.83).     

Autoantibodies directed to novel components of the PM/Scl complex, the human exosome

The autoantigenic polymyositis/scleroderma (PM/Scl) complex was recently shown to be the human homologue of the yeast exosome, which is an RNA-processing complex. Our aim was to assess whether, in addition to targeting the known autoantigens PM/Scl-100 and PM/Scl-75, autoantibodies also target recently identified components of the PM/Scl complex. The prevalence of autoantibodies directed to six novel human exosome components (hRrp4p, hRrp40p, hRrp41p, hRrp42p, hRrp46p, hCsl4p) was determined in sera from patients with idiopathic inflammatory myopathy (n = 48), scleroderma (n = 11), or the PM/Scl overlap syndrome (n = 10). The sera were analyzed by enzyme-linked immunosorbent assays and western blotting using the affinity-purified recombinant proteins. Our results show that each human exosome component is recognized by autoantibodies. The hRrp4p and hRrp42p components were most frequently targeted. The presence of autoantibodies directed to the novel components of the human exosome was correlated with the presence of the anti-PM/Scl-100 autoantibody in the sera of patients with idiopathic inflammatory myopathy (IIM), as was previously found for the anti-PM/Scl-75 autoantibody. Other clear associations between autoantibody activities were not found. These results further support the conception that the autoimmune response may initially be directed to PM/Scl-100, whereas intermolecular epitope spreading may have caused the autoantibody response directed to the associated components.     

Monoclonal antibodies against chicken type V collagen: production, specificity, and use for immunocytochemical localization in embryonic cornea and other organs

Two monoclonal antibodies have been produced against chick type V collagen and shown to be highly specific for separate, conformational dependent determinants within this molecule. When used for immunocytochemical tissue localization, these antibodies show that a major site for the in situ deposition of type V is within the extracellular matrices of many dense connective tissues. In these, however, it is largely in a form unavailable to the antibodies, thus requiring a specific “unmasking” treatment to obtain successful immunocytochemical staining. The specificity of these two IgG antibodies was determined by inhibition ELISA, in which only type V and no other known collagen shows inhibition. In ELISA, mixtures of the two antibodies give an additive binding reaction to the collagen, suggesting that each is against a different antigenic determinant. That both antigenic determinants are conformational dependent, being either in, or closely associated with, the collagen helix is demonstrated by the loss of antibody binding to molecules that have been thermally denatured. The temperature at which this occurs, as assayed by inhibition ELISA, is very similar to that at which the collagen helix melts, as determined by optical rotation. This gives strong additional evidence that the antibodies are directed against the collagen. The antibodies were used for indirect immunofluorescence analyses of cryostat sections of corneas and other organs from 17 to 18-day-old chick embryos. Of all tissues examined only Bowman’s membrane gave a strong staining reaction with cryostat sections of unfixed material. Staining in other areas of the cornea and in other tissues was very light or nonexistent. When, however, sections were pretreated with pepsin dissolved in dilute HAc or, surprisingly, with the dilute HAc itself dramatic new staining by the antibodies was observed in most tissues examined. The staining, which was specific for the anti-type V collagen antibodies, was largely confined to extracellular matrices of dense connective tissues. Experiments using protease inhibitors suggested that the “unmasking” did not involve proteolysis. We do not yet know the mechanism of this unmasking; however, one possibility is that the dilute acid causes swelling or conformational changes in a type-V collagen-containing supramolecular structure. Further studies should allow us to determine whether this is the case.     

New chloramphenicol resistance locus in Bacillus subtilis.

A spontaneously occurring, noninducible, chloramphenicol-resistant mutant of Bacillus subtilis 168 has a mutation (cam-2) which maps in the ribosomal protein region of the chromosome near dal. Its presence does not confer dependence on chloramphenicol. Ribosomes of the cam-2 strain remained sensitive to chloramphenicol in in vitro protein synthesis. No chloramphenicol acetyltransferase activity could be detected.     

Zinc concentration in erythrocyte membranes in normal volunteers and in patients with taste and smell dysfunction

Erythrocyte membrane zinc concentration was measured in 14 normal volunteers and in 36 patients with taste and smell dysfunction, and compared with zinc concentration in erythrocyte cytosol and blood plasma in the normal volunteers and with zinc concentration in erythrocyte cytosol, blood serum, urine, and parotid saliva in the patients. A A significant negative correlation was found between age and membrane zinc concentration in the normals. A significant positive correlation was found between parotid saliva zinc and erythrocyte membrane zinc concentrations in the patients. Concentration of zinc in the erythrocyte membrane is 50% greater than its concentration in cytosol, and three times greater than the zinc concentration in plasma or serum; membrane zinc concentration in humans is approximately one-half that reported in the dog or in the mouse.     

Studies on the mitogenic effect of transferrin by membrane signal transduction

One of the earliest events leading to cell activation and growth is the hydrolysis of inositol phospholipids producing various membrane signals induced by an interaction between growth factors or hormones with their respective receptors on the cell membrane [1].To demonstrate the mitogenic action of transferrin,our results show that an addition of transferrin to “serum-deprived” rat hepatoma cells produced a rapid but transient rise in inositol 1,4,5-trisphosphate(IP3) level,and at the same time,an increased intracellular Ca^2 activity and a cytoplasmic alkalinization were observed.These signal transductions further lend support to the mitogenic nature of transferrin.In addition,a possible link between the receptor-mediated endocytosis of transferrin with the generation of intracellular signals is discussed herewith.     

Equilibrium constants under physiological conditions for the condensation of acetaldehyde with tetrahydrofolic acid

The nonenzymatic reaction of ethanol-derived CH₃CHO with tissue constituents continues to be of interest as a potential mechanism underlying the toxicity of alcohol. The current study has focused on the spontaneous condensation of CH₃CHO with H₄folate under physiological conditions (38 °C, pH 7.0, I = 0.25 M). Computer analysis of uv spectral changes with increasing CH₃CHO concentrations demonstrated the presence of at least two different adducts. The observed equilibrium constant (K_obs) for the formation of the first adduct is 91 ± 2 m^?1 (121 ± 2 m^?1 at 25 °C), a value which is unaffected by variations in ionic strength (0.06–1.0 m) or by free [Mg²⁺] up to 5 mm. The NMR spectrum is compatible with the structure: 5,10-CH₃CH-H₄folate analogous to the naturally occurring 5,10-CH₂-H₄folate. The formation of the latter compound from HCHO and H₄folate, however, is much more favorable under the same conditions [K_obs = 3.0 ± 0.2 × 10⁴ M^?1 (38 °C), 3.6 ± 0.1 × 10⁴ M^?1 (25 °C)]. At the levels of CH₃CHO which accumulate during ethanol metabolism in vivo only a small fraction of the H₄folate will exist as the CH₃CHO derivative, yet it may ultimately be the ratio of free CH₃CHO to free HCHO in tissue which determines the physiological importance of the CH₃CHO adduct. Other adduct(s) of CH₃CHO with H₄folate are observed at very high levels of CH₃CHO but are unlikely to be of physiological significance.