In vitro inhibition of mumps virus by retinoids

Background

Foot-and-mouth disease virus (FMDV) initiates infection via recognition of one of at least four cell-surface integrin molecules α_vβ₁, α_vβ₃, α_vβ₆, or α_vβ₈ by a highly conserved Arg-Gly-Asp (RGD) amino acid sequence motif located in the G-H loop of VP1. Within the animal host, the α_vβ₆ interaction is believed to be the most relevant. Sub-neutralizing levels of soluble secreted α_vβ₆ (ssα_vβ₆) was used as a selective pressure during passages in vitro to explore the plasticity of that interaction.

Results

Genetically stable soluble integrin resistant (SIR) FMDV mutants derived from A24 Cruzeiro were selected after just 3 passages in cell culture in the presence of sub-neutralizing levels of ssα_vβ₆. SIR mutants were characterized by: replication on selective cell lines, plaque morphology, relative sensitivity to ssα_vβ₆ neutralization, relative ability to utilize α_vβ₆ for infection, as well as sequence and structural changes. All SIR mutants maintained an affinity for α_vβ₆. Some developed the ability to attach to cells expressing heparan sulfate (HS) proteoglycan, while others appear to have developed affinity for a still unknown third receptor. Two classes of SIR mutants were selected that were highly or moderately resistant to neutralization by ssα_vβ₆. Highly resistant mutants displayed a G145D substitution (RGD to RDD), while moderately resistant viruses exhibited a L150P/R substitution at the conserved RGD + 4 position. VP1 G-H loop homology models for the A-type SIR mutants illustrated potential structural changes within the integrin-binding motif by these 2 groups of mutations. Treatment of O1 Campos with ssα_vβ₆ resulted in 3 SIR mutants with a positively charged VP3 mutation allowing for HS binding.

Conclusions

These findings illustrate how FMDV particles rapidly gain resistance to soluble receptor prophylactic measures in vitro. Two different serotypes developed distinct capsid mutations to circumvent the presence of sub-neutralizing levels of the soluble cognate receptor, all of which resulted in a modified receptor tropism that expanded the cell types susceptible to FMDV. The identification of some of these adaptive mutations in known FMDV isolates suggests these findings have implications beyond the cell culture system explored in these studies.     

In vitro inhibition of Helicobacter pylori by micromycetes

The anti-Helicobacter pylori effect of 22 micromycetes were studied against one standard strain and 11 clinical isolates of H. pylori. Penicillium ochlochloron and Penicillium funiculosum have been proven the most active fungi against this microorganism. Further bio-guided chemical analysis of P. funiculosum afforded an active component identified as (-) 2,3,4-trihydroxybutanamide.     

Protective effects of Anthocleista djalonensis A. Chev root extracts against induced testicular inflammation and impaired spermatogenesis in adult rats

This study was designed to explore the protective effects of methanol (Meth, 200 mg kg^?1 body wt) and aqueous ethanol (Eth-OH, 200 mg kg^?1 body wt) extracts of Anthocleista djalonensis roots on testicular inflammation induced by lipopolysaccharide (LPS, 5 mg/kg body wt) and depletion of tubular germ cells induced by busulfan (15 mg/kg body wt) in rats after 60 days of oral administration. As expected, LPS stimulation of the animals significantly increased serum and intra-testicular interleukin-6 and serum nitrite levels which were significantly inhibited in the Eth-OH?+?LPS and Meth?+?LPS animals. The increase in testicular and not serum myeloperoxidase activity that was induced by LPS treatment was synergistically increased in the Eth-OH?+?LPS animals, whereas it was inhibited in the Meth?+?LPS animals compared to LPS-treated animals. Furthermore, the administration of the Eth-OH or Meth extracts protected against busulfan-induced depletion of tubular germ cells and promotes the re-population of the seminiferous tubules with germ cells (spermatogonia, spermatocytes and round spermatids) at different stages of development. The extracts were found to contain 7′-oxaspiro [cyclopropane-1,4′-tricyclo [3.3.1.0 (6,8)] nonan-2′-one], cis,cis-7,10-hexadecadienal, hexadecanoic acid, methyl ester, hexadecanoic acid, ethyl ester, 9,12-octadecadienoic acid, methyl ester, and 9,12-octadecadienoic acid (Z,Z)–) which may partly explain the observed anti-inflammatory effects. In conclusion, Meth extracts of A. djanonesis have better anti-inflammatory effects than the Eth-OH extract for the management of impaired testicular function due to inflammation. However both extracts exhibited protective effect on the histology of the testis allowing for the recovery of spermatogenesis.

     

In vitro inhibition of cholinesterases by carbamates--a kinetic study

Kinetics and mechanism of in vitro hydrolyses of acetylcholine and acetylthiocholine by carbamates were studied in a batch reactor at 25 degrees C, pH 8, and ionic strength of 0.11 M. Every hydrolysis was monitored by 3-4 independent methods. All studied hydrolyses can be described by the model of competitive inhibition with an irreversible step (k3). A table of obtained average values of rate constants and discussion of the resultes are given.     

In vitro inhibition of natural-killer-mediated lysis by chromatin fragments

A qualitative impairment of natural killer (NK) function and the presence of circulating DNA have been independently reported in clinical situations such as cancer and lupus. The existence of receptors for chromatin fragments at the leukocyte membrane raised the question of the relation between the presence of chromatin fragments in the extracellular medium and the impairment of NK function. The present study shows that plasmas from patients with metastatic cancer and with pathological DNA concentrations inhibited significantly the NK activity of normal lymphocytes as compared to cancer plasmas with DNA concentrations in the normal range. In vitro, it was demonstrated that chromatin fragments inhibited the NK-mediated cytotoxicity in a dose-dependent manner. Inhibitory concentrations of nucleosomes (2.5–10 g/ml) were lower than those of DNA and histones alone (100 g/ml). Inhibitory effects of nucleosomes, DNA and histones differed also according to the effector population used: nucleosomes were effective whatever the CD56⁺ cell enrichment of the effector population, while DNA inhibition needed T cells, and histone inhibition probably resulted from a subtoxic effect, prevented by the presence of adherent cells. Finally we found that nucleosomes could inhibit the NK function only when they were present in the extracellular medium. Taken together, these data suggest that the persistence of nucleosomal DNA at sites of cell death or in the blood might be responsible, at least partly, for the NK activity impairment observed in pathological circumstances characterized by a high rate of cell death phenomena such as cancer.     

In vitro inhibition of Sphaeropsis sapinea by natural stilbenes

The effects of pinosylvin, pinosylvin monomethyl ether, pinosylvin dimethyl ether, and resveratrol on the fungal shoot blight and canker pathogen of conifers Sphaeropsis sapinea were examined in vitro. Effects of compounds, isolates, and concentrations on both conidial germination and mycelial growth were significant (values of P < 0.001), indicating inhibitory activity of these compounds.     

In vitro inhibition of bacterial growth by iron chelators

The antimicrobial activity of the iron(III)-selective 3-hydroxypyridin-4-one chelators, CP251(1) and CP252(2), was evaluated in comparison with that of diethylenetriamine-penta acetic acid (3). CP251 was found to exhibit an inhibitory effect on the growth of both Gram-positive and Gram-negative bacteria. CP251 may find application in the treatment of external infections such as those associated with wounds.     

In vitro inhibition of mouse dental development by tetracycline

Embryonic molars and incisors were dissected from mandibles of 15-day post-fertilization C57BL/10 mouse embryos and were cultured in vitro for six days on agar-solidified Eagle's basal medium. Experimental explants were cultured on medium which was the same as the control except that 50, 75 or 100 microgram/ml tetracycline was added. Treated explants of both incisors and molars were suppressed in development and reduced in size. Enamel organs and dental papillae of all tooth germs subjected to higher tetracycline concentrations were abnormal in structure and differentiation of ameloblasts and odontoblasts was inhibited. Explants treated with higher dosage levels of the drug were more severely affected than those exposed to lower concentrations. Recovery from the suppression induced by tetracycline was observed in explants transferred to control medium for four days of growth following treatment. Differentiated ameloblasts and odontoblasts observed in the recovering tooth germs indicated that the inhibition in development was temporary. The results of this study showed that tetracycline can alter dental development in vitro prior to mineralization. The observed inhibition may be related to a disruption of collagen biosynthesis which is thought to play a role in the controlling epithelial-mesenchymal interaction involved in tooth germ morphogenesis.     

In vitro acetylcholinesterase inhibition by type A botulinum toxin

Type A botulinum toxin was studied for its ability to inhibit the action of acetyl-cholinesterase. The chromogenic substrate, indophenyl acetate, was used for assay of enzyme activity. Inhibition of enzyme function was detected through use of both 6.6 x 10(-6) mg (20 ld(50)) and 6.6 x 10(-10) mg (2 x 10(-3)ld(50)) of type A botulinal toxin. Control assays were performed by use of both homologous antitoxin and heterologous antitoxins (types B and E). Enzyme inhibition was effectively prevented by use of homologous antitoxin only. The inhibition noted was specific and reproducible for given substrate, enzyme, and toxin concentrations.     

In vitro inhibition of Helicobacter pylori by extracts of thyme

Extracts of several plants were tested for inhibitory activity against Helicobacter pylori. Among these plants thyme (aqueous extract) and cinnamon (alcoholic extract) were the most effective. Since aqueous extract of thyme is easier to produce and consume, it was further investigated. Compared with several antibacterials, the thyme extract had a significant inhibitory effect on H. pylori , reducing both its growth and potent urease activity. From the results of this study, the aqueous extract of thyme possesses a therapeutic potential which merits validation by clinical studies.     

In vitro inhibition of aromatase by the enantiomers of aminoglutethimide and analogs

The in vitro aromatase activity in microsomal fractions from rat ovary and its inhibition by enantiomers of aminoglutethimide (AG), rogletimide (RG), and cyclohexylaminoglutethimide (ChAG) were studied by analysing the [³H]H₂O released when [1β-³H]androstenedione was converted to estrone. Maximum velocity (V_max) and the Michaelis-Menten constant (K_m) of the microsomal aromatase enzyme were 17.40 ± 0.45 pmol/ml/mg protein/min and 1.02 ± 0.06 μM, respectively. The IC₅₀s for the enantiomers were similar for (+)-R-AG and (?)-R-ChAG (0.86 ± 0.06 and 0.89 ± 0.15 μM, respectively). (+)S-ChA'G was most potent with IC₅₀ of 0.075 ± 0.003 μM. The IC₅₀s for (?)-S-AG, (+)-R-RG, and (?)-S-RG were in the same range (23.15 ± 2.74, 24.58 ± 2.46, and 24.43 ± 2.20 μM, respectively). © 1994 Wiley-Liss, Inc.     

In vitro inhibition by estrogens of the oxidative modifications of human lipoproteins

Estrogens exert protective actions against atherosclerosis, part of these effects having been ascribed to their antioxidant properties. The aim of this work was to assess the ability of estrogens to prevent the oxidative modifications of low density lipoproteins (LDL) and other plasma lipoprotein fractions whose relationship with atherosclerosis has been less studied. For this purpose, different estrogen compounds were used: natural and synthetic estrogens, and catecholestrogens. The molecules were added in vitro to human LDL and very low density lipoproteins (VLDL) in the presence of Cu2+. The lipoprotein oxidative modifications were determined by measuring the formation of thiobarbituric acid reactive substances, the appearance of conjugated dienes and the degradation of tryptophan groups from the apoproteins. In VLDL, 2-hydroxyestradiol and diethylstilbestrol exerted potent antioxidant effects similar to those found for alpha-tocopherol and probucol. 17beta-Estradiol and 4-hydroxyestradiol also prevented VLDL oxidation, but to a lesser extent. When LDL were used, estrogens similarly exerted antioxidant actions, 2-hydroxyestradiol being the most potent inhibitor. These results show that estrogens, whose antioxidant actions have been demonstrated in other experimental models, also possess the ability to prevent in vitro the oxidative modifications of human plasma LDL and VLDL.