Bernard F. Driscoll

Damage to Neurons in Culture Following Medium Change: Role of Glutamine and Extracellular Generation of Glutamate

Abstract— Changing the medium of primary cell cultures of CNS origin causes severe damage that is mediated via the N‐methyl‐d‐aspartate (NMDA)‐type of glutamate receptors and dependent on the presence of glutamine in the medium. Data presented here show that glutamine has two roles in culture damage: glutamine is contaminated with a small amount of glutamate, which is responsible for initiating culture damage, and glutamine is the source of the glutamate that is produced extracellularly in damaged cultures. The NMDA receptor plays a critical role minutes after medium change when the glutamate contaminating the glutamine binds to NMDA receptors; during this time, addition of a low level (10–20 μM) of 2‐amino‐5‐phos‐phonovaleric acid can block most culture damage and the appearance of extracellular glutamate. A higher level (300 μM) of 2‐amino‐5‐phosphonovaleric acid can protect cultures when added at much later times (30–60 min). Between 3 and 6 h after medium change, the concentration of extracellular glutamate starts to rise and accumulates until the end of the culture period (20 h). Medium removed from cultures at 3 h or later after medium change and incubated alone (i.e., with no cells) also continues to generate glutamate; filtration (0.22 μrn pore size) or centrifugation (18,000 g) stops the appearance of this glutamate. 6‐Diazo‐5‐oxo‐l‐norleucine, an inhibitor of the mitochondrial enzyme glutaminase, blocks the generation of glutamate. Mitochondria or mitochondrial fragments are probably released from the damaged cells and then convert extracellular glutamine to glutamate, resulting in generation of a high extracellular glutamate concentration.

Myelin Basic Protein: Location of Multiple Independent Antigenic Regions

Immnunization of guinea pigs with homologous myelin basic protein induces antibodies that differ in their ability to bind specific peptide fragments of the protein. Antiserums with differing specificities made it possible to demonstrate at least three mutually exclusive antigenic sites in the protein molecule. One of these sites is located between residues 44 and 89, another between 90 and 116, and the third between 117 and 170.

Cell Damage Associated with Changing the Medium of Mesencephalic Cultures in Serum-Free Medium Is Mediated via N-Methyl-D-Aspartate Receptors

Abstract: Dopaminergic neurons from embryonic rat mesencephalon were grown in simple serum‐free media. The cells develop over a period of several weeks in vitro, particularly between day 14 and day 23. Removing the culture medium and replacing it with fresh medium during this interval caused severe damage to the cultures; this damage is mediated by excitatory amino acids acting through glutamate receptors. Damage could be completely prevented by antagonists of the N‐methyl‐D‐aspartate subtype of glutamate receptor. As expected, medium that contains glutamate (i.e., Hams F‐12 medium) caused damage; however, medium that contains no glutamate or aspartate (i.e., Dulbeccos modified Eagle medium) also caused severe damage, and most of the damage was dependent on the presence of glutamine in the medium. The presence of the antibiotics penicillin and streptomycin greatly enhanced damage caused by medium change.

IMMUNOCHEMICAL AND BIOCHEMICAL STUDIES DEMONSTRATING THE IDENTITY OF A BOVINE SPINAL CORD PROTEIN (SCP) AND A BASIC PROTEIN OF BOVINE PERIPHERAL MYELIN (BF)

A protein extracted from bovine peripheral myelin (BF) and a protein extracted from bovine spinal cord (SCP) have been shown to be identical: the proteins cross‐react immunochemicaliy with each other but not with highly purified CNS myelin basic protein. Neither BF nor SCP have anti‐encephalitogenic activity. Their electrophoretic behavior is the same at three different pH values. Their apparent molecular weight by sodium dodecyl sulfate‐gel electrophoresis is 13,800 ± 550. The amino acid compositions of the proteins are essentially identical. BF and SCP each contain 2 cysteine residues and have valine at the C terminus. The 23 major tryptic peptides are identical on peptide maps. Circular dichroic analyses yield essentially identical curves, which, when computed by best‐fit curve analysis, indicate that each has 0%α helix and a large percentage of β structure.

LPS augments adoptive transfer of experimental allergic encephalomyelitis in the Lewis rat.

Adoptive transfer of experimental allergic encephalomyelitis (EAE) is enhanced after in vitro culture of myelin basic protein (BP)-sensitized lymphoid cells with BP. Addition of lipopolysaccharide (LPS) to the culture further augments transfer of EAE to a level 5 times greater than that achieved with cells activated only with BP. Neither the proliferative response of a BP-specific cell line nor the production of IL-2 by BP-sensitized lymphoid cells in response to BP was augmented by the addition of LPS to the culture. Augmentation of EAE was also observed if recipients received simultaneous injections of BP-sensitized lymph node cells (BP/LNC) cultured with BP (BP-activated) and normal spleen cells cultured independently with LPS (LPS/Spl-C). To analyze the effect of contact between these two cell populations in vivo, we mixed the two cell populations in vitro at reduced cell concentrations. When BP-activated BP-LNC were mixed with LPS-Spl-C in vitro, a marked synergistic proliferative response was observed. Irradiation of BP-activated BP/LNC abrogated this synergistic response, whereas irradiation of LPS/Spl-C did not, suggesting that the proliferating population was in the BP/LNC and that the LPS/Spl-C enhanced their proliferation. These results indicate that LPS exerts its effect through BP-nonspecific cells and that these cells enhance transfer of EAE by augmenting the proliferation of the BP-specific cells in vivo after transfer.

Mechanism of demyelination in the guinea pig. Separate sensitization with encephalitogenic myelin basic protein and nonencephalitogenic brain components.

Experimental allergic encephalomyelitis (EAE), accompanied by demyelinating central nervous system (CNS) lesions, can be induced in guinea pigs sensitized with whole guinea pig CNS tissue, but not in animals sensitized with purified myelin basic protein (BP). This type of chronic demyelinating EAE is presumably a result of a combination of a cell-mediated immune response to the encephalitogenic BP and a separate response to other nonencephalitogenic CNS antigens. We report here that demyelinating EAE can be induced when separate sensitizations are used to induce a cell-mediated response to BP and a second immune response to nonencephalitogenic CNS antigens. Animals sensitized in separate sites with guinea pig BP and whole chicken brain develop CNS demyelinating lesions. Animals sensitized only to BP or chicken brain do not develop demyelination.

Severity of demyelination in vivo correlates with serum myelination inhibition activity in guinea pigs having a new form of experimental allergic encephalomyelitis

Guinea pigs received a suboptimal transfer of lymphocytes sensitized to myelin basic protein (BP) and were then immunized with guinea pig BP, BP plus chicken brain or chicken myelin, or chicken brain alone. Sera from these animals were tested for the presence of myelinotoxic antibodies, as detected by the myelination inhibition assay. Myelination inhibition activity correlated with the histologic severity of demyelination.