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%.     

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.     

The hot IVGTT two-compartment minimal model: an improved version

The two-compartment minimal model (2CMM) interpretation of a labeled intravenous glucose tolerance test (IVGTT) is a powerful tool to assess glucose metabolism in a single individual. It has been reported that a derived 2CMM parameter describing the proportional effect of glucose on insulin-independent glucose disposal can take physiologically unplausible negative values. In addition, precision of 2CMM parameter estimates is sometimes not satisfactory. Here we resolve the above issues by presenting an improved version of 2CMM that relies on a new assumption on the constant component R(d0) of insulin-independent glucose disposal. Here R(d0) is not fixed to 1 mg x kg(-1) x min(-1) but instead is expressed as a fraction of steady-state glucose disposal. The new 2CMM is identified on the same stable labeled IVGTT data base on which the original 2CMM was formulated. A more reliable insulin-independent glucose disposal portrait is obtained while that of insulin action remains unchanged. The new 2CMM also improves the precision with which model parameters and metabolic indexes are estimated.     

Toxoplasma gondii: an improved rat model of congenital infection

The objective of this study was to refine the rat model of congenital toxoplasmosis. In Fischer rats we found that visualization of spermatozoa in vaginal exudates and the detection of at least 6 g body weight increase between days 9 and 12 of pregnancy, allowed the diagnosis and timing of pregnancy with 60% specificity and 84% sensitivity. A dose of 10⁴Toxoplasma gondii bradyzoites or 10²T. gondii oocysts of the Prugniaud strain resulted in more than 50% of congenital infection of the rat litters. Transmission of T. gondii via lactation was not detected in rats inoculated with either bradyzoites or oocysts. Bioassays of 51 neonates born from mothers inoculated with bradyzoites (in tissue cysts) and 29 neonates from mothers inoculated with oocysts demonstrated that both liver and lungs can be used for the diagnosis of congenital transmission in this model.     

Use of caryophyllene oxide as an antifungal agent in an in vitro experimental model of onychomycosis

Caryophyllene oxide, an oxygenated terpenoid, well known as preservative in food, drugs and cosmetics, has been tested in vitro as an antifungal against dermatophytes. Its antifungal activity has been compared to ciclopiroxolamine and sulconazole, commonly used in onychomycosis treatment and chosen because of their very different chemical structures. So, a new model has been tested, utilizing sheep hoof plates in order to simulate human nails, which are almost unobtainable for in vitro tests. Three protocols were utilized: pre-treatment. simultaneous treatment and post-treatment. Among these, the post-treatment method was the best to simulate antifungal therapy. as it permitted testing and comparing the efficiency of different antifungal drugs.This revised version was published online in October 2005 with corrections to the Cover Date.     

In vitro culture: an epigenetic challenge for plants

In vitro plant cell and tissue culture techniques are the basis of many micropropagation and breeding programs for scientific research. Plant tissue culture (PTC) involves organogenesis and embryogenesis, and the outcome depends on the different conditions to which the tissue is exposed. PTC is a stressful environment—high relative humidity, low ventilation rate, high concentrations of plant growth regulators, and low light availability—for plants that need to rapidly change their molecular regulation in order to respond fast and efficiently during cell division and growth. For instance, somatic embryogenesis (SE), which plays an important role in plant multiplication, requires complex cellular, biochemical and molecular processes for embryo formation and development. New data has come out about a connection between plant morphogenesis and epigenetics. Epigenetics is a very sensitive regulatory mechanism, which in most of cases is affected by the environment. Although it is known that, under plant morphogenesis, the genome has little or no change, DNA methylation and histone modifications are very susceptible to those in vitro environmental conditions. In the present review, we highlight the most used in vitro systems such as organogenesis and SE in plants and discuss how epigenetics plays a pivotal role in the phenotype outcome. Furthermore, we discuss the big role that the small RNAs have during cell division and propagation and propose different challenges and opportunities to study epigenetics in plant cell tissue and organ cultures.