Antagonism against fomes annosus. Comparison between different test methods in vitro and in vivo

The antagonism of Trichoderma harzianum, Scytalidium album and an unidentified fungus (isolated from Monotropa hypopitys) against the root rot fungus Fomes annosus was studied in vitro and in vivo. Antibiosis was demonstrated on malt extract agar and on powdered wood. Competition between F. annosus and its antagonists was studied in stem discs and in living stems of Norway spruce. Effective antibiotics on agar and sawdust did not always bring about effective antagonism against F. annosus in stem discs or living trees. Factors such as growth rate of the antagonists in wood and their ability to invade the substrate during competition may influence the effectiveness of F. annosus suppression. The results from studies on antagonism in stem discs agreed with the results from tests in living trees. The frequency of penetrations through the wood cell walls was drastically lowered by the presence of the antagonists but was not closely correlated with the abundance of F. annosus.     

Comparison between different rabbit antisera against the glucocorticoid receptor

Seven antisera against the glucocorticoid receptor (GR), raised in different rabbits immunized with highly purified (in case of five rabbits apparently homogeneous) preparation of GR from rat liver cytosol, were compared concerning titer and cross-reactivity. The titers of protein A-purified antisera (10 mg/ml) were in the range 1:100-1:320 as measured by enzyme-linked immunosorbent assay, ELISA, (defined as the dilution giving 50% of maximum absorbance). All seven antisera bound to the rat GR with a Stokes radius of 6.1 nm, but no antiserum reacted with the proteolytically induced steroid binding domain with Stokes radius 3 nm. However, the antigenic determinant(s) of the non-ligand-binding domain(s), split off from the steroid binding domain, is preserved following digestion with alpha-chymotrypsin or trypsin, respectively, since immunoactivity is still detectable by ELISA. Only two of four antisera tested cross-reacted with the GR from human lymphocytes. The same two antisera cross-reacted with chick embryo liver GR. Four out of four antisera tested cross-reacted with mouse liver GR as well as with rabbit lung GR. For these antisera, antibody binding to the GR prior to steroid- or DNA-binding did not influence the ability of the GR to interact with the ligand or DNA-cellulose, respectively. No difference regarding avidity of the antisera for activated or non-activated GR was observed. Furthermore, none of the antisera tested cross-reacted with the estrogen, progestin, androgen or mineralocorticoid receptors in rat. These findings indicate that the antisera from different rabbits raised against the same antigen all react with a certain domain of the rat GR, but show species differences as well as receptor class specificity.     

Biochemical and functional changes during immunization of rats with angiotensin II

Angiotensin-converting enzyme (ACE) activity in serum and some brain areas, level of angiotensin I in the blood and drinking behaviour during immunization of rats against conjugate of angiotensin II with bovine serum albumin (BSA) were studied. The results show that an increase in antibodies against angiotensin II was correlated with elevated ACE activity in serum. There was a distinct tendency towards elevated level of angiotensin I in the blood. After a 6 month's immunization ACE-activity was reduced twofold to threefold in midbrain and hypothalamus-thalamus. During immunization water-uptake was increased by 40-45%.     

Production of antibodies against the porcine luteinizing hormone by two immunization methods.

Two methods of rabbit immunization were applied: multisite intradermal injections of small doses and intramuscular injections of large hormone doses for obtaining antibodies against the porcine luteinizing hormone (LH). A high titre, specificity towards LH and affinity were obtained in rabbits immunized by both methods. In most animals the highest titre was noted 10 weeks after the first immunization.     

Prophylactic immunization against experimental leishmaniasis. VI. Comparison of protective and disease-promoting T cells

In previous studies, we reported that mice immunized i.v. with lethally irradiated Leishmania major promastigotes developed substantial resistance to a subsequent L. major infection. However, such protection could be totally suppressed by prior s.c. injection with the same antigens. Both the protective immunity and the inhibition of its induction could be adoptively transferred with specific Lyt-2- T cells. Here, we present evidence showing that protection and disease promotion resulting from i.v. or s.c. immunization, respectively, are mediated by functionally distinct subsets of T cells. In a series of titration experiments, it was found that freshly isolated T cells derived from prophylactically i.v. immunized BALB/c mice were either protective (greater than 10(7) cells/recipient) or ineffective (less than 10(7) cells/recipient). No exacerbation of disease was observed at any dose. Conversely, T cells from mice immunized s.c. either accelerated disease development and inhibited protective immunization (greater than 10(7) cells/recipient) or had no effect (less than 10(7) cells/recipient). No protection was observed at any dose tested. In mixed transfer experiments, increasing numbers of T cells from s.c. immunized donors progressively inhibited the protective effect of T cells from i.v. immunized donors. Supernatant of T cell cultures from protectively immunized donors contained substantial macrophage-activating factor whereas such activity was not detectable in the supernatant of T cell culture from s.c. immunized donors. Analysis by flow cytometry showed that the spleen and lymph nodes of normal, i.v., or s.c. immunized BALB/c mice contained similar ratios of L3T4+ cells and Lyt-2+ cells.     

Ergosterol determination in Saccharomyces cerevisiae. Comparison of different methods

Several methods for ergosterol determination in two different Saccharomyces cerevisiae strains were tested. The best results were obtained by a simple method including saponification with 8 M KOH, extraction of non-saponifiable lipids and ergosterol determination by reversed phase HPLC. The methods in which the samples were treated with 0.1 M HCl prior to hydrolysis with 2.5 or 4.5 M water-ethanolic KOH underestimated the ergosterol contents.     

Comparison of three gamma-turn mimetic scaffolds incorporated into angiotensin II

Rigidification of peptides by cyclization and iterative incorporation of well-defined secondary structure mimetics constitutes one approach to the design of non-peptidergic structures with better defined conformations. We herein present the synthesis of a potential gamma-turn mimetic scaffold, and its incorporation in the 3-5 position of angiotensin II. Two analogues of angiotensin II (Ang II) incorporating this 1,3,5-trisubstituted benzene gamma-turn scaffold were synthesized. Evaluation of the compounds in a radioligand binding assay showed that they lacked affinity to the AT1 receptor. To rationalize these results a geometrical and electrostatical comparison with Ang II analogues encompassing a bicyclic scaffold that delivered inactive pseudo peptides and an azepine scaffold producing highly active ligands was made. This analysis did not provide a clear rationale for the inactivity of the benzene gamma-turn scaffolds.     

Comparison of the solution structures of angiotensin I & II. Implication for structure-function relationship.

Conformational analysis of angiotensin I (AI) and II (AII) peptides has been performed through 2D 1H-NMR spectroscopy in dimethylsulfoxide and 2,2,2-trifluoroethanol/H2O. The solution structural models of AI and AII have been determined in dimethylsulfoxide using NOE distance and 3JHNHalpha coupling constants. Finally, the AI family of models resulting from restrained energy minimization (REM) refinement, exhibits pairwise rmsd values for the family ensemble 0.26 +/- 0.13 A, 1.05 +/- 0.23 A, for backbone and heavy atoms, respectively, and the distance penalty function is calculated at 0.075 +/- 0.006 A2. Comparable results have been afforded for AII ensemble (rmsd values 0.30 +/- 0.22 A, 1.38 +/- 0.48 A for backbone and heavy atoms, respectively; distance penalty function is 0.029 +/- 0.003 A2). The two peptides demonstrate similar N-terminal and different C-terminal conformation as a consequence of the presence/absence of the His9-Leu10 dipeptide, which plays an important role in the different biological function of the two peptides. Other conformational variations focused on the side-chain orientation of aromatic residues, which constitute a biologically relevant hydrophobic core and whose inter-residue contacts are strong in dimethylsulfoxide and are retained even in mixed organic-aqueous media. Detailed analysis of the peptide structural features attempts to elucidate the conformational role of the C-terminal dipeptide to the different binding affinity of AI and AII towards the AT1 receptor and sets the basis for understanding the factors that might govern free- or bound-depended AII structural differentiation.     

Characteristics of physiological reactions during long-term immunization of rats with protein conjugates of angiotensin II

The results show that during long-term immunization of rats against conjugates of angiotensin II with bovine serum albumin definite age dynamics of immune response have been observed. It was shown that the changes of physiological readings were correlated with growing of antibodies titer against angiotensin II, accordingly, as the immune response was lower, these readings return to the initial level.