Regulation of cyclic adenosine 3',5'- monophosphate signaling and pulsatile neurosecretion by Gi-coupled plasma membrane estrogen receptors in immortalized gonadotrophin-releasing hormone neurons

Immortalized GnRH neurons (GT1-7) express receptors for estrogen [estrogen receptor-alpha and-13(ERa and ERI3)] and progesterone (progesterone receptor A) and exhibit positive immunostaining for both intracellular and plasma membrane ERs. Exposure of GT1-7 cells to picomolar estradiol concentrations for 5-60 min caused rapid, sustained,and dose-dependent inhibition of cAMP production. In contrast, treatment with nanomolar estradiol concentrations for 60 min increased cAMP production. The inhibitory and stimulatory actions of estradiol on cAMP formation were abolished by the ER antagonist, ICI 182,780. The estradiol-induced inhibition of cAMP production was prevented by treatment with pertussis toxin, consistent with coupling of the plasma membrane ER to an inhibitory G protein. Coimmunoprecipitation studies demonstrated an estradiol-regulated stimulatory interaction between ERa and G,3 that was prevented by the ER antagonist, ICI 182,780. Exposure of perifused GT1-7 cells and hypothalamic neurons to picomolar estradiol levels increased the GnRH peak interval, shortened peak duration, and increased peak amplitude. These findings indicate that occupancy of the plasma membrane-associated ERs expressed in GT1-7 neurons by physio-logical estradiol levels causes activation of a G, protein and modulates cAMP signaling and neuropeptide secretion.     

Multigene DNA prime-boost vaccines for SHIV89.6P

We assessed four prime-boost vaccine regimens with a Gene Gun component for SHIV89.6P in Macaca nemestrina. A dosing experiment using beta-galactosidase plasmid showed that 30 or 45 shots per dose elicited higher titer antibody than smaller doses. For SHIV89.6P, we administered a six-plasmid vaccine capable of producing non-infectious virions in vivo in combination with either vaccinia recombinants or inactivated virus. DNA prime/vaccinia boost, or the reverse, elicited strong immune responses. The SHIV89.6P challenge virus was grown in M. nemestrina peripheral blood mononuclear cells and titered in vivo intrarectally. As has been observed for SHIV89.6P in M. mulatta, the infected M. nemestrina experienced rapid and severe loss of circulating CD4+ T cells. Vaccinated macaques were challenged three weeks after the last boost. DNA prime/vaccina boost or vaccina prime/DNA boost protected 11/12 animals from acute CD4+ T cell depletion and disease, while other regimens were not effective.     

Development of a fluorescence immunoassay for measurement of paclitaxel in human plasma

A new fluorescence immunoassay for the quantitative determination of paclitaxel (Pac) under equilibrium conditions was developed. Anti-Pac IgG2a antibody was immobilized through its Fc region to protein A covalently bound to the inside surface of a silanized glass capillary column and the antigen-binding sites of anti-Pac saturated with rhodamine-labeled Pac (Rh-Pac). Analyte Pac was circulated through the column in a closed loop and the steady-state fluorescence of the Rh-Pac displaced from the immobilized antibody was recorded after 6 min. The Rh-Pac fluorescence emission intensity was directly related to the concentration of the Pac analyte over a broad dynamic range of up to 400 ng/ml with a linear range up to 200 ng/ml and lower detection limit of 5.85 ng/ml. While there was no interference from the baccatin III and 10-deacetylbaccatin III, cephalomannine was found to interfere in Pac determination. When applied for measurement of Pac in human plasma, the concentration of Pac determined by the fluorescence assay was found to be in excellent agreement with the Pac added, confirming the potential of the fluorescence immunoassay for clinical application.     

High-affinity anti-ganglioside IgG antibodies raised in complex ganglioside knockout mice: reexamination of GD1a immunolocalization

Gangliosides, sialic acid-bearing glycosphingolipids, are highly enriched in the vertebrate nervous system. Anti-ganglioside antibodies are associated with various human neuropathies, although the pathogenicity of these antibodies remains unproven. Testing the pathogenic role of anti-ganglioside antibodies will be facilitated by developing high-affinity IgG-class complement-fixing monoclonal anti-bodies against major brain gangliosides, a goal that has been difficult to achieve. In this study, mice lacking complex gangliosides were used as immune-naive hosts to raise anti-ganglioside antibodies. Wild-type mice and knockout mice with a disrupted gene for GM2/GD2 synthase (UDP-N-acetyl-D-galactosamine : GM3/GD3 N-acetyl-D-glactosaminyltransferase) were immunized with GD1a conjugated to keyhole limpet hemocyanin. The knockout mice produced a vigorous anti-GD1a IgG response, whereas wildtype littermates failed to do so. Fusion of spleen cells from an immunized knockout mouse with myeloma cells yielded numerous IgG anti-GD1a antibody-producing colonies. Ganglioside binding studies revealed two specificity classes; one colony representing each class was cloned and characterized. High-affinity monoclonal antibody was produced by each hybridoma : an IgG1 that bound nearly exclusively to GD1a and an IgG2b that bound GD1a, GT1b, and GT1aalpha. Both antibodies readily readily detected gangliosides via ELISA, TLC immune overlay, immunohistochemistry, and immunocytochemistry. In contrast to prior reports using anti-GD1a and anti-GT1b IgM class monoclonal antibodies, the new antibodies bound avidly to granule neurons in brain tissue sections and cell cultures. Mice lacking complex gangliosides are improved hosts for raising high-affinity, high-titer anti-ganglioside IgG antibodies for probing for the distribution and physiology of gangliosides and the pathophysiology of anti-ganglioside antibodies.