In vitro neurotransmitter release in an animal model of depression.

Sprague-Dawley rats exposed to uncontrollable shock can be separated by a subsequent shock escape test into two groups: a "helpless" (LH) group which demonstrates a deficit in escape behavior, and a "nonlearned helpless" (NLH) group which shows no escape deficit and acquires the escape response as readily as naive control rats (NC) do. The present studies were designed to examine the correlations between the behavioral differences and the changes of in vitro neurotransmitter release seen in these three groups of rats. The major finding concerned a significant increase in endogenous and K(+)-stimulated serotonin (5-HT) release in the hippocampal slices of LH rats. There were no apparent differences in acetylcholine, dopamine and noradrenaline release in the hippocampus of LH rats as compared to NLH and NC rats. These results add further support to previous studies in our laboratory which implicate presynaptic 5-HT mechanisms in the behavioral deficit caused by uncontrollable shock.     

Neuropathy in experimental diabetes: an animal model.

A morphometric study of the common peroneal nerve in early experimental diabetes in rats showed that fibre size was diminished. The reduction in the size of the axon was twice that of the myelin sheath. This may contribute to the understanding of the impaired motor conduction velocity found in diabetics shortly after the onset of their disease.     

Immobilized heparinase: In vitro reactor model

Heparinase immobilized to agarose has previously been shown to be useful in degrading heparin and thereby preventing thromboembolytic complications when this anticoagulant has been used in extracorporeal perfusions. The current study examined the kinetics of this immobilized enzyme. When heparinase is covalently bound to 8% agarose, the partition coefficient of heparin in the catalytic particle is 0.36 +/- 0.048 (N = 10). The immobilized enzyme has a K(m) of 0.15 +/- 0.03 mg/mL and an activation energy of 10.3 +/- 0.57 kcal/gmol (N = 5). These values are statistically indistinguishable from the values for the free enzyme. The immobilized enzyme showed a pH activity optimum between 7.0 and 7.4, compared to the optimum pH of 6.5 for the soluble enzyme. The activity optimum of immobilized heparinase with respect to salt concentration was between 0 and 0.1M. A reactor containing immobilized heparinase recirculating internally at 1300 mL/min behaved as a continuously stirred tank reactor (CSTR) when solutions at a flow rate of 120 mL/min were passed through the device. The residence time distribution was determined using blue dextran (molecular weight 2 x 10(6) daltons), which is sterically excluded from the agarose catalyst. A model of the heparinase reactor based on ideal CSTR behavior and the immobilized enzyme kinetic parameters was developed. It accurately predicted experimental conversions over a range of catalyst volumes, enzyme loadings, and substrate concentrations to within 7% in most cases and with a maximum deviation of 13%.     

In vitro delayed hypersensitivity granuloma formation: development of an antigen-coated bead model

Previously, we have described an in vitro model of granulomatous hypersensitivity around Schistosoma mansoni eggs in both the murine model of schistosomiasis and in human schistosomiasis. These studies describe a new model of in vitro granuloma formation that complexes soluble egg antigen from S. mansoni eggs, a partially purified protein derivative of Mycobacterium tuberculosis (PPD), or bovine serum albumin to carrier beads. Ultrastructural and morphologic evaluations demonstrate that there are initial macrophage interactions, followed by the recruitment of antigen-specific T cells that interact with and recruit macrophages, lymphocytes, granulocytes, and fibroblasts. Finally, there is a stage of granulomatous organization involving fibroblast proliferation and collagen deposition. The in vitro reactivity, defined by a quantitative granuloma index, correlates with in vivo granulomas around S. mansoni eggs in the livers of infected cell donor animals. In vitro granuloma formation against PPD-coated beads correlated with delayed cutaneous hypersensitivity against PPD, which was judged by footpad swelling. The reactions demonstrate antigenic specificity and were intrinsically modulated in a manner that is analogous to that previously shown with the in vitro egg granuloma model. This model of in vitro granuloma formation promises to be a useful tool for elucidating mechanisms of cellular immunity and regulation.     

In vitro transformation of petunia cells by an improved method of co-cultivation with A. tumefaciens strains

Summary A method (termed co-cultivation) for transforming plant cells in vitro with A. tumefaciens strains, which was originally developed by Marton et al. (1978) Nature 277: 129–131, has been modified by the incorporation of a novel feeder plate culture system and been extended to use with petunia protoplasts. Using efficient cell plating and selection conditions for phytohormone-independent growth, large numbers of independent transformed calli can be obtained efficiently (10^-1) and in less than 3 weeks following protoplast isolation. Southern hybridization analysis has confirmed that the majority of the resulting in vitro transformants contain a single copy of full length T-DNA.The high efficiency of this procedure allows simple screening to identify plant cells transformed by Ti plasmids attenuated by deletion of internal T-DNA regions. Results are presented that demonstrate the co-cultivation method can be used in conjunction with short term assays for monitoring plant gene expression.     

Virus-neuron interaction: an experimental model

Conclusion These examples demonstrate the usefulness of embryonic neurons for investigation ofneurotropic virus pathogenesis studies. The different steps of viral interactions of neurotropicviruses with the brain necessitate studies of the intrinsic neural properties of the infected cells:1) entry of virus into the intra-cellular compartment, 2) transport of virus from one brainstructure to another brain area, 3) replication of virus in the host-cells, 4) altered brainfunctions. Understanding the mechanisms of neural pathogenesis is a prerequisite for theelaboration of antiviral strategies based upon recovery of the brain from functional alterationsusing drugs active on brain functions.     

In vitro model for studying malignancy associated changes.

Malignancy associated changes (MAC) can be defined as subtle morphological and physiologic changes that are found in ostensibly normal cells of patients harboring malignant disease. It has been postulated that MAC have a potential to become a useful tool in detection, diagnosis and prognosis of malignant diseases. An in vitro cell culture model system was designed to study interactions between non-small cell lung cancer (NSCLC) and the normal bronchial epithelium of the human respiratory tract in vivo to see if the MAC-like phenomenon can be detected in such a system. In this study we examined changes in nuclear features of normal human bronchial epithelial cells (NHBE) when they were co-cultured with cells derived from a lung cancer cell line NCI-H460. Using discriminant function analysis, nuclear features were determined which allow maximal discrimination between normal cells incubated with or without cancerous cells. Our results demonstrate that MAC appear to be specific to changes induced by malignancy, and that these changes differ from those induced by growth factors in the serum. This study provides evidence in support to the hypothesis that MAC are induced by a soluble factor(s) released by malignant cells. Colour figure can be viewed on http://www.esacp.org/acp/2003/25-2/sun.htm     

Developing an improved In vitro propagation system for slow-growing species using garcinia mangostana L. (mangosteen)

Summary This investigation disclosed that evaluation of tissue culture parameters of slowly developing species (e.g. Garcinia mangostana) requires monitoring of treatments through two or more successive, relatively long passages. Two 8-wk passages were necessary to observe differences in phytohormone effects. Photoperiod and temperature effects were not clearly evident until tissues had been cultured through three passages; the optimal photoperiod and temperature for shoot proliferation could not be established until after the fifth passage. Our investigation revealed that no auxin supplementation was necessary for bud primordium differentiation in cotyledon explants or proliferation of regenerated shoots. The optimum N⁶-benzyladenine concentration for primordium differentiation was 13.3 μM, and for shoot proliferation ranged from 4.4 to 13.3 μM. Continuous culturing in an 8-h photoperiod at 30°C resulted in progressively intensified degeneration of shoots after three passages. In contrast, successive passages in a 16-h photoperiod/26°C regimen enabled sustained regeneration of shoots. The shoots rooted at a rate of 85% when precultured for 3 d in a medium containing 4921.3 μM indole-3-butyric acid, or 10 d at 492.1 μM, then cultured for two 8-wk passages in phytohormone-free medium. Following acclimatization by gradually lowering the relative humidity in the growth chamber, rooted shoots survived transfer to the greenhouse at a rate of 95%.     

In vitro selection of allosteric ribozymes: theory and experimental validation

In vitro selection techniques offer powerful and versatile methods to isolate nucleic acid sequences with specific activities from huge libraries. We describe an in vitro selection strategy for the de novo selection of allosteric self-cleaving ribozymes responding to pefloxacin and other quinolone derivatives. Within 16 selection cycles, highly sensitive clones responding to drug levels in the sub-micromolar range were obtained. The morpholine moiety of the quinolone derivatives was required for inhibition of the self-cleavage of the selected ribozymes: modifications of the aromatic system were tolerated better than modifications of the morpholine ring. We also present a theoretical model that analyzes the predicted fraction of ribozymes with a given binding constant and cleavage rate recovered after each selection cycle. This model precisely predicts the actual experimental values obtained with the selection procedure. It can thus be used to determine the optimal conditions for an in vitro selection of an allosteric ribozyme with a desired dissociation constant and cleavage rate for a given application.