In vitro colonization ability of human colon mucosa by exogenous Lactobacillus strains

Abstract A 5.4 kb Hind III DNA fragment carrying the gene encoding raw starch-digesting α-amylase (RSDA), has been previously cloned from Bacillus circulans F-2 and expressed in Escherichia coli [Kim et al. (1990) Biochim. Biophys. Acta 1048, 2233–2238]. Interestingly, when the cell extract of E. coli harboring a plasmid carrying this fragment was incubated with l M NaCl, it exhibited about 10 times higher enzyme activity than when assayed without NaCl. Differential zymograms showed two different amylase activities: one for RSDA and the other for a salt-dependent a-amylase (SDA). Even though RSDA activity was detected without NaCl, SDA activity was detected only in high concentrations of NaCl. SDA activity was fully detected at above l M NaCl. Results from subcloning of the genes, fractionation analysis of cell extracts, and immunological assays clearly suggested that the two amylases are genetically distinct and that genes for both enzymes are closely linked on the 5.4 kb DNA fragment.     

The proposed 5′-nucleotidase inhibitor in human leukemic cells is an artefact

It is now well established that human lymphoblastoid cell lines showing immaturity characters display ecto-5′-nucleotidase activities lower than normal levels. A recent paper (Sun, A.S., Holland, J.F. and Ohnuma, T. (1983) Biochim. Biophys. Acta 762, 577–584) mentioned that this phenomenon resulted from the presence of a 5′-nucleotidase inhibitor in these cell lines. We demonstrate here that the use of 5′-[³H]AMP as a substrate, and inadequate analysis of the products formed, led them to a misinterpretation. [³H]Adenosine derived from 5′-[³H]AMP hydrolysis was further transformed into [³H]inosine by the adenosine deaminase activity of the leukemic cell lines tested; [³H]inosine was precipitated with the excess substrate and was not taken into account in the ecto-5′-nucleotidase determination, which led the authors to confuse this adenosine deaminase activity with a 5′-nucleotidase inhibitor. We did not observe 5′-nucleotidase inhibition by leukemic cell cytosol when convenient assay methods were used and showed that the presence of such an inhibitor remains to be established.     

Lipoxygenase- and cyclooxygenase-reaction products and incorporation into glycerolipids of radiolabeled arachidonic acid in the bovine retina

The metabolism of radiolabeled arachidonic acid (AA) by the intact bovine retina has been studied. Synthesis of prostaglandins (PGs) and hydroxyeicosatetraenoic acids (HETEs), and incorporation of AA into glycerolipids has been measured by reverse-phase and straight-phase high performance liquid chromatography with flow scintillation detection, and by thin-layer chromatography. AA was actively acylated into glycerolipids, particularly triglycerides, phosphatidylcholine and phosphatidylinositol. AA was also converted to the major PGs, PGF_2α, PGE₂, PGD₂, 6-keto-PGF_1α and TXB₂, and to the lipoxygenase reaction products, 12-HETE, 5-HETE, and other monohydroxy isomers. Approximately 6% of the radiolabeled AA was converted to eicosanoids. The synthesis of HETEs was inhibited in a concentration-dependent manner (IC₅₀ = 8.3 NM) by nordihydroguaiaretic acid (NDGA). PG synthesis was inhibited by aspirin (10 μM), indomethacin (1 μM) and NDGA (IC₅₀ = 380 nM). Metabolism of AA via lipoxygenase, cyclooxygenase and activation-acylation was inhibited by boiling retinal tissue prior to incubation. These studies demonstrate an active system for the uptake and utilization of AA in the bovine retina, and provide the first evidence of lipoxygenase-mediated metabolism of AA, resulting in the synthesis of mono-hydroxyeicosatetraenoic acids, in the retina.     

Arachidonic acid-induced human platelet aggregation independent of cyclooxygenase and lipoxygenase

It is generally agreed that arachidonic acid (20:4ω6) can stimulate platelet aggregation after conversion to prostaglandin G₂ and H₂ and thence to thromboxane A₂. This action is prevented by cyclooxygenase inhibitors. Washed platelets were isolated on metrizamide gradient and resuspended in a Ca²⁺-free buffer. Their stimulation by C 20:4 6 was followed by ¹⁴C serotonin (5HT) release, thromboxane (TX) synthesis and an increase of light transmission, not dependent on aggregation, accompanied by slight lysis (14%). The addition of extrinsic Ca²⁺ suppressed lysis and allowed the formation of aggregates. Under these conditions, cyclooxygenase inhibitors such as acetyl salicylic acid, indomethacin or flurbiprofen totally suppressed TX synthesis without preventing platelet aggregation or [¹⁴C]-5HT release. Other C 20 polyunsaturated fatty acids could not substituted for C 20:4ω6 in inducing aggregation, and Ca²⁺ was found to be a prerequesite for protection of the cell against lysis as well as for aggregation in the absence or TX formation. The use fo the lipoxygenase inhibitor BW 755 C did not prevent C 20:4ω6-induced aggregation of aspirin-treated platelets, suggesting that the phenomenon was independent of this pathway also. The total suppression of oxidative metabolism with these inhibitors was verified by the analysis of icosanoids using glass capillary column gas chromatography. It is suggested that under these condition, C 20:4ω6-induced platelet aggregation might be due to an increased membrane permeability to Ca²⁺ induced by this fatty acid in the absence of oxidation.     

Isolation of methotrexate-resistant cell lines in Petunia hybrida upon stepwise selection procedure

Summary Cell suspensions of Petunia hybrida were subjected to a selection procedure in which the concentration of the selective agent, methotrexate (MTX), was gradually elevated. In mammalian cells, this procedure frequently results in MTX-resistant mutants due to amplification of the gene coding for dihydrofolate reductase (DHFR), the target protein of MTX.Five suspension lines were isolated, with degrees of resistance ranging from 10 to 500 M MTX (in wild type the LD_99.9 is 0.2 M). MTX^R phenotypes were unstable, as manifested by the loss of resistance upon prolonged growth in the absence of drug. All of the mutants also exhibited high values of MTX-binding protein (60- to 400-fold higher than that of the wild type), which declined to intermediate values upon MTX withdrawal. Finally, cellular extracts from all of the mutants also showed high specific staining of DHFR-activity in gels.The results suggest that the resistance of MTX in these plant cell-lines is mediated by the elevation of the amounts of DHFR, probably as a consequence of gene amplification.     

Nonsense RNA: a tool for specifically inhibiting the expression of a gene in vivo

We describe a general technique to inhibit gene expression in eukaryotic cells. The gene we chose to inhibit was the E. coli LacZ gene (encoding beta-galactosidase), which has previously been cloned into a eukaryotic expression vector [1]. This plasmid is called pCH110. We constructed a variant of pCH110 in which we flipped a 2566 base pair 5' fragment of the LacZ gene into the antiparallel orientation. The plasmid containing this mutated LacZ gene is called pNSLacZ (NS signifies non-sense coding sequence). When equal amounts of pCH110 and pNSLacZ are co-transfected into 3T6 mouse fibroblasts, the beta-galactosidase activity is decreased by approximately a factor of ten. Increasing the ratio of pNSLacZ to pCH110 above 1:1 does not appreciably increase the level of inhibition. Next, we prove the specificity of the inhibition by adding a third gene to the transfection mixture. For this purpose, we used pSVneo beta, a plasmid which expresses a phosphotransferase. We found that even when the beta-galactosidase activity was diminished by a factor of 10, the phosphotransferase activity was unaffected. Therefore, we have demonstrated that: the presence of an antiparallel copy of the LacZ gene results in a significant and specific diminution of the LacZ gene's expression; only a fraction of the LacZ gene needs to be in the antiparallel orientation in order to observe this effect. These results suggest that this technique can serve as a tool to decrease the level of gene expression in order to study the function of specific genes, or as a therapeutic manoeuvre in the treatment of disorders of abnormal gene expression.     

Innervation du muscle rétracteur antérieur du byssus (ABRM) de Mytilus edulis L. et de Mytilus galloprovincialis Lmk.

Summary Specific and non specific cholinesterase activities were demonstrated in the ABRM of Mytilus edulis L. and Mytilus galloprovincialis L. by means of different techniques. The results were found identical for both species: neuromuscular junctions en grappe-type scarcely distributed within the ABRM, contain AChE.According to the histochemical inhibition tests, (a) the eserine inhibits AChE activity of the ABRM with a level of 5·10^–5 M or higher, (b) the ChE non specific activities are inhibited by iso-OMPA level between 5·10^–5 to 10^–4 M.The histo- and cytochemical observations were completed by showing the existence of neuromuscular junctions containing small clear vesicles: they probably are the morphological support for ACh presence.Moreover, specific and non specific ChE activities were localized in the glio-interstitial cells. AChE precipitates were developped along the ABRM sarcolemma, some muscle mitochondria and in the intercellular spaces remain enigmatic.

---

---

L'auteur dédicace cette contribution à son père, Mr. Anthime Gilloteaux à l'occasion de son 75ème anniversaire     

Histological and ultrastructural studies of Beauveria bassiana infection in Leptinotarsa decemlineta larvae during ecdysis

Studies of Leptinotarsa decemlineata larvae infected by Beauveria bassiana during ecdysis have enabled us to define the modes of fungal penetration employed to enter the ecdysial cuticle. We have observed the mechanically active passage of the penetrant hyphae and have followed the growth of the filaments and blastospore formation in the molting fluid. The attack of the new integument and its consequent alteration and the entry into the body cavity have also been studied. The infection develops rapidly in some of the larvae which die in premolt, while others are able to molt. Conditions rendered abnormal due to the presence of the fungus cause integumentary injuries which serve as an important factor in pathogenesis since they enhance the entry of fungal elements and bacteria thereby inducing septicemia. Contaminated larvae are able to molt, showing no signs of injury or disease, and survive for a long time, until the fungus finally invades the organism and causes death. This postponement of mortality shows that molting and hemocytic reactions are, to a certain extent, an effective defense mechanism. These last observations can be useful in the understanding of pathological processes associated with a hidden phase of fungal infection.