Methods for rapid screening and isolation of bacteria producing acidic lipase: feasibility studies and novel activity assay protocols

Acidic lipase finds its commercial values in medical applications and bioremediation of food wastes. In this work, approaches for rapid screening of lipase-producing bacteria were developed and the feasibility assessment of the screening methods was performed. From food waste samples, the proposed screening procedures allowed isolation of sixteen pure bacterial strains expressing higher lipase activity at acidic pH (pH 6.0) than at alkaline pH (pH 9.0). To enhance the accuracy of lipase activity determination under acidic conditions, a novel assay procedure was also developed by deactivating lipase activity by microwave treatment prior to back titration. This additional step could minimize interferences arising from residual lipase activity during conventional direct back-titration methods in measuring lipase activity at acidic pH. Using the four strategies proposed in this work, the best acidic-lipase-producing isolate was obtained by strategy C (SSC) and was identified as Aeromonas sp. C14, displaying an optimal lipase activity of 0.7 U/ml at an acidic pH of 6.0.     

Heliothrix oregonensis,gen. nov., sp. nov., a phototrophic filamentous gliding bacterium containing bacteriochlorophyll a

An unusual filamentous, gliding bacterium was found in a few hot springs in Oregon where it formed a nearly unispecific top layer of microbial mats. It contained a bacteriochlorophyll a-like pigment and an abundance of carotenoids. There were no chlorosomes or additional chlorophylls. The organism was aerotolerant and appeared to be photoheterotrophic. It was successfully co-cultured with an aerobic chemoheterotroph in a medium containing glucose and casamino acids. Although it has many characteristics in common with the genus Chloroflexus, the lack of chlorosomes and bacteriochlorophyll c and the aerobic nature of this organism indicate that it should be placed in a new genus. This conclusion is supported by 5S rRNA nucleotide sequence data.     

Fine structure of early human embryos frozen with 1,2 propanediol

Previous studies by a French group (Fertil Steril 44:645–651, 1985) have shown that two-to eight-cell human embryos can survive slow freeze-thawing with propanediol in a biological freezer. These embryos were assessed for morphological appearance by phase-contrast microscopy. We assessed the structure of 25 frozen-thawed one- to 12-cell embryos, obtained from our in vitro fertilization (IVF) and GIFT programmes, by phase-contrast and electron microscopy, using the same method of cryopreservation. One-fourth of the embryos examined had all cells intact, and more than one-half the embryos had over 50% of their cells well preserved. Some of these embryos had unequal blastomeres and cytoplasmic fragments. Ultrastructural assessment revealed good preservation of fine structure in the intact blastomeres of all embryos and maintenance of cell-to-cell contacts. Most cytoplasmic organelles, cell membranes, and nuclei were well preserved compared to nonfrozen controls. The cells that were cryoinjured showed varying degrees of disorganization of the cell membrane, cytosol, and cellular membranes, including swelling and disruption of the nuclear envelope. Disruption of the zona was somewhat rare. Small cytoplasmic fragments were less prone to cryoinjury than blastomeres. The use of propanediol for embryo cryopreservation seems to be feasible; frozen embryos with more than 50% cells intact have produced 10 pregnancies after embryo transfer (Fertil Steril 46:268–272, 1986). Replacement of 17 frozen embryos in seven patients has resulted in a twin pregnancy in Singapore. However, the effects of freezing on the mitotic spindles of embryonic cells need to be investigated further.     

Comparison of the relative sensitivity of human lymphocytes and mouse splenocytes to two spindle poisons

Aneugenic compounds act on non-DNA targets to exert genotoxicity via an indirect mechanism. In contrast to DNA-binding agents, these compounds are expected to possess threshold levels of activity. Therefore, the risk for adverse effects following human exposure to an aneugen could be minimal, if the threshold of activity has been clearly determined in vivo and in vitro and providing the human exposure level is below this threshold. Thus, the development of a single-cell model to allow comparisons between in vitro and in vivo threshold values for aneugenic compounds is of importance.The in vivo micronucleus test is one of the main assays used in genetic toxicology, and is often performed in the mouse. Thus, an extensive database is available in the literature. However, there are only few data concerning the in vitro micronucleus assay using mouse cells, as the majority of in vitro micronucleus assays have been performed using human lymphocytes. In addition, there is a lack of data concerning thresholds for any compound using this model.First, we evaluated whether the use of mouse splenocytes would be an acceptable alternative to that of human lymphocytes to identify aneugens. To allow valid comparisons, the two protocols were first harmonized. Thus, phytohemagglutinin (PHA) and concanavalin A were used as specific mitogens for human lymphocytes and mouse splenocytes, respectively, in order to achieve similar cell-proliferation rates. To achieve similar and sufficient numbers of binucleated cells, cytochalasin B was added 44 and 56 h after culture initiation of the human and mouse cells, respectively.Second, we compared the sensitivity of the mouse protocol with that of the human protocol by exposing the cells to the aneugens nocodazole and paclitaxel.There was good reproducibility of the cytotoxic/genotoxic responses of the two cell models following exposure to the aneugens. The sensitivity of the mouse splenocytes to paclitaxel was higher than that of the human lymphocytes. The two cell types were equally sensitive to nocodazole.     

Relative performance of monohaploid potato clones and their diploid parents at plant level and after protoplast isolation and subsequent fusion

Summary Plant growth performance was studied in 118 potato monohaploids and in their diploid parents. Of these monohaploids 76 were also investigated at the protoplast level and eight of these were used in protoplast fusion experiments as well. No correlation was found between relative performance of greenhouse grown and in vitro grown plants. No or only weak correlations were found between different in vitro characteristics such as plant growth, protoplast yield per gram plant material, plating efficiency and callus growth. This indicates the unpredictability of these characters.The protoplast fusion experiments indicated that only in some genotype combinations increased callus growth rates may be found. However, it is not clear whether such calli were hybrids or not. In protoplast monocultures only diploid and tetraploid regenerants were obtained. After fusion, tetraploids but also some triploids could be regenerated. The finding of triploids indicates that monoploid protoplasts were involved in fusion. Isozyme analysis and morphological assessment of the plants pointed out that the majority of the fusion regenerants were hybrids. The implications of these results are discussed.     

A novel array-based assay of in situ tissue transglutaminase activity in human umbilical vein endothelial cells

Transglutaminases (TGs), a family of calcium-dependent transamidating enzymes, are involved in functions such as apoptosis and inflammation and play a role in autoimmune diseases and neurodegenerative disorders. In this study, we describe a novel array-based approach to rapidly determine in situ TG activity in human umbilical vein endothelial cells and J82 human bladder carcinoma cells. Amine arrays were fabricated by immobilizing 3-aminopropyltrimethoxysilane on glass slides. The assay was specific and highly reproducible. The average coefficient of variation betweens spots was 2.6% (n = 3 arrays), and the average correlation coefficients between arrays and between arrays/reactions were 0.998 and 0.976, respectively (n = 3 arrays). The assay was successfully applied to detect changes in TG activity induced by maitotoxin and to analyze inhibition of the TG activation with cystamine and monodansyl cadaverine. In addition, the assay demonstrated that intracellular reactive oxygen species regulate the maitotoxin-induced activation of TG. Thus, the array-based in situ TG activity assay constitutes a rapid and high-throughput approach to investigating the roles of TGs in cell signaling.     

In vitro reactivation of sarin-inhibited brain acetylcholinesterase from different species by various oximes

In vitro as well as in vivo evaluation of the reactivating efficacy of various oximes against nerve agent-inhibited acetylcholinesterase has been usually done with the help of animal experiments. Nevertheless, previously published data indicate that the reactivation potency of oximes may be different in human and animal species, which may hamper the extrapolation of animal data to human data. Therefore, to better evaluate the efficacy of various oximes (pralidoxime, obidoxime, HI-6, K033) to reactivate brain acetylcholinesterase inhibited by sarin by in vitro methods, human, rat and pig brain acetylcholinesterase were used to calculate kinetic parameters for the reactivation. Our results show differences among the species, depending on the type of oxime, and indicate that data from animal experiments needs to be carefully evaluated before extrapolation to humans.     

Pre-exposure affects the olfactory response of Drosophila melanogaster to menthol

A modification of the trap assay (Woodard et al., 1989) was used to evaluate the response of Drosophila melanogaster (Meigen) to food media containing menthol. Dose-response curves for flies to mentholic foods were produced for flies that had been pre-exposed to menthol, during development and adult life, and flies that had not been exposed to menthol before the assay. Mentholic food media were less attractive to Drosophila than plain food medium. Rearing flies on a medium containing menthol reduced their aversion to some concentrations of menthol. The rearing effect was not simply due to lowered general activity levels resulting from developing in a medium containing menthol. There was a threshold concentration of menthol in the rearing medium below which we found no induced behavioural change.     

Elimination of apple stem grooving virus by chemotherapy and development of an immunocapture RT-PCR for rapid sensitive screening

Ombuin (7,4′-dimethyl quercetin) (10 μg ml^-1, for 12 wk), glycyrrhizin/quercetin (80 μg ml^-1and 10 μg ml^-1respectively, for 18 wk), ribavirin (10 μg ml^-1, for 12 wk) and quercetin/ribavirin (10 μg ml^-1each, for 9–12 wk) reduced the titre of apple stem grooving virus (ASGV) when applied in vitro to infected tissue cultures of Nicotiana occidentalis obliqua Wheeler, and/or Malus domestica. ASGV was not detectable in both plant species after the quercitin/ribavirin treatment when tested by ISEM, herbaceous host indexing, RT-PCR, and immunocapture RT-PCR. A sensitive immunocapture RT-PCR procedure for the detection of ASGV was developed for the screening of treated samples to assess antiviral activity.