Neuronal interactions during epileptic events in vitro

Epileptic events can be produced in in vitro brain slices after perfusion with convulsant agents such as penicillin or picrotoxin. These events consist of one or more synchronized neuronal bursts. In this experimental system, epileptic events occur because of blockade of synaptic inhibition by the convulsant agent. A sparse network of excitatory synaptic interconnections in the hippocampus serves to synchronize a population of neurons, each of which is capable of bursting after appropriate stimulation.     

Tumour cell interactions in vitro

After addition of dibutyryl cAMP to spinner flask cultures of JB-1-E tumour cells an increase in the number of examples of emperipolesis was observed. Ruthenium red staining of the cells revealed a conspicuous staining of the cell membrane of the outer cell, while that of the inner cell was unstained. The differences in staining properties of the cell membranes of the outer and inner cells involved in emperipolesis and apparently viable inner cells make it evident that coexistence of cells--one within another--is possible.     

In vitro cultivation of Wolbachia in insect and mammalian cell lines

Wolbachia infecting the small brown planthopper, Laodelphax striatellus, were successfully maintained and cultivated in two insect and one mammalian cell lines. The bacteria with the planthopper ovary were introduced into the flasks with the cultures of the cell lines. The Wolbachia proliferated in mosquito (Aedes albopictus) and lepidopteran (Heliothis zea) cell lines and in the mouse cell line, L929. Proliferation of Wolbachia was confirmed by electron microscopy and quantitative polymerase chain reaction. This simple method for the cultivation of Wolbachia was applicable to other strains of Wolbachia, such as the one found in the lepidopteran eggs, and should facilitate fundamental and applied studies of this important group of microorganisms.     

Antioxidant defense systems of two lipidopteran insect cell lines

Spodoptera frugiperda Sf-9 (Sf-9) and Trichoplusia ni BTI-Tn-5B1-4 (Tn-5B1-4) insect cell lines were found to contain unique assemblages of antioxidant enzymes. Specifically, the Sf-9 insect cell line contained Manganese and Copper-Zinc superoxide dismutase (MnSOD and CuZnSOD) for reducing the superoxide radical (O(2)(*-)) to hydrogen peroxide (H(2)O(2)) and ascorbate peroxidase (APOX) for reducing the resulting H(2)O(2) to H(2)O. Approximately one third of the total SOD activity was found to be MnSOD. The Tn-5B1-4 cells were also found to contain MnSOD (approximately two thirds of the total SOD activity), CuZnSOD and APOX activities. However, the Tn-5B1-4 cell line, in contrast to the Sf-9 cell line, contained catalase (CAT) activity for reducing H(2)O(2) to H(2)O. Both the Sf-9 and Tn-5B1-4 cell lines contained glutathione reductase and dehydroascorbic acid reductase activities for regenerating the reduced forms of glutathione and ascorbic acid, respectively. In addition, both cell lines contained glutathione S-transferase peroxidase activity towards hydroperoxides other than H(2)O(2). Finally, neither cell line contains the glutathione peroxidase activity that is ubiquitous in mammalian cells.     

Expression of functional recombinant antibody molecules in insect cell expression systems

Recombinant single-chain variable-fragment molecules (scFv) were constructed from a cell line expressing a monoclonal antibody against African cassava mosaic virus (ACMV) and expressed in Escherichia coli. DNA sequences that encoded the scFv were manipulated to allow scFv expression in insect cell lines. A recombinant baculovirus containing the scFv cDNA was constructed and large amounts of scFv were produced in each of three insect cell lines infected with the baculovirus. However, the scFv were not secreted into the medium by any of the cell lines despite the scFv having been linked to a honeybee melittin leader sequence. The same scFv cDNA construct was introduced into Drosophila DS2 cells and a stable recombinant cell line was obtained that produced scFv that was secreted into the medium. Culture medium containing the scFv was used directly in enzyme-linked immunosorbent assay (ELISA) tests to detect ACMV in plant tissues. Another construct that encoded the Ckappa domain of human IgG was fused to the C-terminus of the scFv that was produced and expressed in Drosophila cells. This scFv derivative also accumulated in the medium and was more active in ELISA than scFv lacking the Ckappa domain.     

Purification of replication factors using insect and mammalian cell expression systems

Purification of factors for DNA replication in an amount sufficient for detailed biochemical characterization is essential to elucidating its mechanisms. Insect cell expression systems are commonly used for purification of the factors proven to be difficult to deal with in bacteria. We describe first the detailed protocols for purification of mammalian Mcm complexes including the Mcm2/3/4/5/6/7 heterohexamer expressed in insect cells. We then describe a convenient and economical system in which large-sized proteins and multi-factor complexes can be transiently overexpressed in human 293T cells and be rapidly purified in a large quantity. We describe various expression vectors and detailed methods for transfection and purification of various replication factors which have been difficult to obtain in a sufficient amount in other systems. Availability of efficient methods to overproduce and purify the proteins that have been challenging would facilitate the enzymatic analyses of the processes of DNA replication.     

Brain lipid binding protein in axon-Schwann cell interactions and peripheral nerve tumorigenesis

Loss of axonal contact characterizes Schwann cells in benign and malignant peripheral nerve sheath tumors (MPNST) from neurofibromatosis type 1 (NF1) patients. Tumor Schwann cells demonstrate NF1 mutations, elevated Ras activity, and aberrant epidermal growth factor receptor (EGFR) expression. Using cDNA microarrays, we found that brain lipid binding protein (BLBP) is elevated in an EGFR-positive subpopulation of Nf1 mutant mouse Schwann cells (Nf1(-/-) TXF) that grows away from axons; BLBP expression was not affected by farnesyltransferase inhibitor, an inhibitor of H-Ras. BLBP was also detected in EGFR-positive cell lines derived from Nf1:p53 double mutant mice and human MPNST. BLBP expression was induced in normal Schwann cells following transfection with EGFR but not H-Ras12V. Furthermore, EGFR-mediated BLBP expression was not inhibited by dominant-negative H-Ras, indicating that BLBP expression is downstream of Ras-independent EGFR signaling. BLBP-blocking antibodies enabled process outgrowth from Nf1(-/-) TXF cells and restored interaction with axons, without affecting cell proliferation or migration. Following injury, BLBP expression was induced in normal sciatic nerves when nonmyelinating Schwann cells remodeled their processes. These data suggest that BLBP, stimulated by Ras-independent pathways, regulates Schwann cell-axon interactions in normal peripheral nerve and peripheral nerve tumors.     

Neuronal signaling systems and ethanol dependence

In recent years there have been remarkable developments toward the understanding of the molecular and/or cellular changes in the neuronal second-messenger pathways during ethanol dependence. In general, it is believed that the cyclic adenosine 3′, 5′-monophosphate (cAMP) and the phosphoinositide (PI) signal-transduction pathways may be the intracellular targets that mediate the action of ethanol and ultimately contribute to the molecular events involved in the development of ethanol tolerance and dependence. Several laboratories have demonstrated that acute ethanol exposure increases, whereas protracted ethanol exposure decreases, agonist-stimulated adenylate cyclase activity in a variety of cell systems, including the rodent brain. Recent studies indicate that various postreceptor events of the cAMP signal transduction cascade (i.e., G_s protein, protein kinase A [PKA], and cAMP-responsive element binding protein [CREB]) in the rodent brain are also modulated by chronic ethanol exposure. The PI signal-transduction cascade represents another important second-messenger system that is modulated by both acute and chronic ethanol exposure in a variety of cell systems. It has been shown that protracted ethanol exposure significantly decreases phospholipase C (PLC) activity in the cerebral cortex of mice and rats. The decreased PLC activity during chronic ethanol exposure may be caused by a decrease in the protein levels of the PLC-Β₁ isozyme but not of PLC-δ₁ or PLC-γ₁ isozymes in the rat cerebral cortex. Protein kinase C (PKC), which is a key step in the Pi-signaling cascade, has been shown to be altered in a variety of cell systems by acute or chronic ethanol exposure. It appears from the literature that PKC plays an important role in the modulation of the function of various neurotransmitter receptors (e.g., γ-aminobutyrate type A [GABAa], N-methyl-D-aspartate [NMDA], serotonin2A [5-HT2a], and 5-HT2C, and muscarinic [m1] receptors) resulting from ethanol exposure. The findings described in this review article indicate that neuronal-signaling proteins represent a molecular locus for the action of ethanol and are possibly involved in the neuroadaptational mechanisms to protracted ethanol exposure. These findings support the notion that alterations in the cAMP and the PI-signaling cascades during chronic ethanol exposure could be the critical molecular events associated with the development of ethanol dependence.     

Plant polyploidy and insect/plant interactions

We used flow cytometry and extensive geographic surveys of herbivore attack to test whether repeated evolution of autotetraploidy in the perennial herb Heuchera grossulariifolia Rydb. (Saxifragaceae) has created evolutionary barriers to attack by the specialist moth herbivore Greya politella (Prodoxidae). We found that the moth has colonized tetraploid as well as diploid populations, has colonized tetraploids of separate evolutionary origin, and, at least under some conditions, is more likely to attack tetraploids than diploids. Plant polyploidy therefore provides a potential route out of specialization as an evolutionary dead end in phytophagous insect taxa as well as a potentially important route to subsequent phylogenetic and geographic diversification of plant/insect interactions.     

Neuronal and glial cell biology

Here, we review progress in our understanding of neuronal and glial cell biology during the past ten years, with an emphasis on glial cell fate specification, apoptosis, the cytoskeleton, neuronal polarity, synaptic vesicle recycling and targeting, regulation of the cytoskeleton by extracellular signals, and neuron-glia interactions.     

Antigen-specific. I region-restricted interactions in vitro between tumor cell lines and T cell hybridomas

A series of H-2d B cell tumor lines and one monocytic tumor cell line were shown to be capable of I region-restricted antigen presentation to I-A-d- and I-Ed- restricted, antigen-specific cloned T cell hybridomas. For the most part, antigen presentation correlated with the present of Ia antigens on the presenting cells, although in a few interesting cases Ia-expression lines failed to present antigen. These T cell hybridomas, together with the B cell and to monocyte tumor cell lines, offer a unique set of tools to study the phenomenon of I region-restricted antigen presentation.