The Drosophila abrupt gene encodes a BTB-zinc finger regulatory protein that controls the specificity of neuromuscular connections.

Motor axons make synaptic connections with specific muscles, and this specificity unfolds during development as motoneuron growth cones make specific pathway choices and ultimately recognize and synapse on their specific muscle targets. The Drosophila clueless mutation was identified previously in a genetic screen for mutations that disrupt motoneuron guidance and connectivity. We show here that clueless is allelic to abrupt. The abrupt gene is required for the embryonic formation of specific synaptic connections between a subset of motoneurons and a subset of muscles. Mutations in abrupt also reveal its role in establishing and maintaining muscle attachments, adult sensory cell formation, and morphogenesis of adult appendages. The abrupt gene encodes a zinc finger protein with a conserved BTB domain. Abrupt is expressed in muscle nuclei but not motoneurons, suggesting that abrupt controls the muscle expression of molecules required for correct motoneuron targeting, as well as molecules required for correct muscle attachments.

Morphological and molecular response of the MOCH-1 oligodendrocyte cell line to serum and interferon-gamma: possible implications for demyelinating disorders.

The regional loss of oligodendrocytes is thought to be an important pathological event in a variety of demyelinating diseases of the central nervous system (CNS). Various components of serum, which are normally excluded from the CNS by the blood-brain barrier, have been implicated as mediators of demyelinating disorders. We have examined the effects of high concentrations of serum (10% fetal bovine serum, FBS), as well as the cytokine interferon-gamma (IFN-gamma), on an oligodendrocyte cell line, MOCH-1 cells. These cells changed from phase-bright, small round cells with multiple thin, branched processes in 1% FBS medium to flat, fibroblast-like cells with large cell bodies when cultured in 10% FBS medium or 1% FBS medium containing IFN-gamma. These morphological changes were accompanied by a large increase in expression of the astrocyte marker, glial fibrillary acidic protein (GFAP), as detected by Northern and Western blot analyses. In addition, Northern blot and fluorescence-activated cell sorting analyses revealed that IFN-gamma induced a very large increase in major histocompatibility complex (MHC) class I expression in MOCH-1 cells. MHC class II mRNA induction by IFN-gamma was also seen. In contrast, 10% FBS did not elevate either MHC class I or class II mRNA levels in MOCH-1 cells. The morphological and molecular effects of 10% FBS and IFN-gamma were reversible. We suggest that the response of MOCH-1 cells to high concentrations of serum and IFN-gamma may reflect an important in vivo response to oligodendrocytes to perturbations that occur in demyelinating disorders.

A rapid and efficient method for the radiosynthesis and purification of [125I]SCH23982.

The radiosynthesis of (1R)-(+)-1-phenyl-3-methyl-7-[125I]iodo-8-hydroxy- 2,3,4,5-tetrahydro-1H-3-benzazepine (commonly referred to as SCH23982) and its use as a high affinity D1 dopamine antagonist ligand have been reported previously. We now provide a simple and inexpensive protocol for the rapid and efficient synthesis of this radioligand based on the Cloramine-T-catalyzed reaction between the commercially available precursor (R)-(+)-1-phenyl-3-methyl- 8-hydroxy-2,3,4,5-tetrahydro-1H-3-benzazepine and carrier-free sodium [125I]iodide. [125I]SCH23982 is separated rapidly (within 20 min) from the precursor and reaction byproducts (e.g., chlorinated precursor, SCH23390) by reverse-phase HPLC on a C-8 column. The major iodinated product has been identified as SCH23982 based on co-chromatography with authentic SCH23982, UV spectral characteristics, and biological activity. The chromatographic effluent containing the active product is adsorbed on a C-18 Sep-Pak cartridge to remove mobile-phase constituents and permit it to be eluted and diluted to the desired concentration; this technique is used also for periodic repurification. Our synthesis protocol results in final purified product that incorporates ca. 50% of the initial 125I (tested using starting quantities of 1-10 mCi Na125I); the final product has a specific activity of ca. 2500 +/- 350 Ci/mmol. Data from in vitro receptor autoradiographic and homogenate studies with this radioligand are consistent with previously reported values in terms of expected receptor distribution, affinity, and density (KD of 1.0 nM, Bmax of 1400 fmol/mg protein in rat striatal membranes).

Neural cell adhesion molecules modulate tyrosine phosphorylation of tubulin in nerve growth cone membranes.

Triggering neural cell adhesion molecules of the immunoglobulin superfamily with specific ligands or antibodies inhibited the phosphorylation of tryosyl residues in a subpopulation of alpha- and beta-tubulin associated with membranes from a subcellular fraction of nerve growth cones from fetal rat brain. Preincubation of these membranes with purified extracellular fragments of L1, N-CAM, or myelin-associated glycoprotein, or with antibodies directed against the extracellular domains of L1 or N-CAM, inhibited pp60c-src-dependent phosphorylation of tubulin in an endogenous membrane kinase reaction. Other proteins that affect neurite outgrowth (fibronectin, laminin, antibodies against N-cadherin) had no effect. The results suggest that cell adhesion molecules transduce cell surface events to intracellular signals by modulating the activity of protein tyrosine kinases or phosphatases in axonal membranes to influence cytoskeletal dynamics at the growth cone.

Catalytic role of histidine 147 in Escherichia coli thymidylate synthase.

Nine mutant thymidylate synthases were isolated that only differed in sequence at position 147. The wild-type enzyme (which had a histidine residue at 147) and mutant enzymes were purified to near homogeneity and their kinetic properties were compared. Although the kcat values for the mutant enzymes were 10-10,000-fold lower than for the wild-type enzyme, the Km values for both 2'-deoxyuridylate and 5,10-methylenetetrahydrofolate were nearly identical for all the enzymes indicating that His-147 is not significantly involved in initial substrate binding. By comparing the wild-type (His-147) to the glycine (Gly-147) enzyme, the side chain of His-147 was estimated to lower the activation energy of the catalytic step by 1.6-2.9 kcal mol-1. In contrast to the wild-type enzyme, the activity of the Gly-147 enzyme decreased when the pH was raised above 7.5. The activity loss coincided with the deprotonation of a residue that had a pKa of 9.46 +/- 0.2 and an enthalpy of ionization (delta Hion) of 12.1 +/- 0.9. These values are consistent with the involvement of a lysine or an arginine residue in the catalytic process. An inspection of the rates of ternary complex formation among enzyme, 5-fluoro-2'-deoxyuridylate, and 5,10-methylenetetrahydrofolate for the mutant enzymes indicated that His-147 is not needed for the proton removal from C-5 of 2'-deoxyuridylate but rather participates in an initial catalytic step and alters the pKa value of a catalytically important lysine or arginine residue.