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Glutamate receptors play critical roles in normal and pathological processes. We developed an antisense gene delivery strategy to modulate the NMDA type of glutamate receptor. Using transient transfection in vitro and viral mediated gene transfer in vitro and in vivo, the effect of expression of an antisense gene fragment (60 bp) of the NR1 subunit was tested. Immunoblot analysis showed an antisense-concentration-dependent reduction in the NR1 subunit upon transient co-transfection of a plasmid expressing a sense NR1 gene and a plasmid expressing the antisense fragment into COS-7 cells. After recombination into an adenoviral vector, this antisense fragment reduced the amount of endogenous NR1 protein in PC12 cells. Finally, direct intraparenchymal injection of the viral vector into rat spinal cord resulted in diminished NR1 in motor neurons. Our results demonstrate the efficacy of this approach, which combines antisense with viral gene delivery to control the expression of specific genes in vivo. This approach may also be useful in reducing excitatory neurotransmission in vivo, with implications for the treatment of spinal disorders such as amyotrophic lateral sclerosis or chronic pain.

Two cDNAs encoding PAK kinases were isolated from a mouse embryo library by screening with a PCR-generated probe derived from the kinase domain of a rat PAK kinase. These cDNAs, designated PAK-1 and PAK-3, encode mouse PAK kinases of 545 and 544 amino acids, respectively. Both proteins possess an N-terminal Cdc42/Rac interacting binding domain (CRIB) and a C-terminal serine/threonine kinase domain. Comparison of the two mouse PAK kinases revealed that the proteins show 87% amino acid identity. Northern analysis of a multiple mouse tissue blot with a PAK-1 probe detected a 3.0kb transcript that was almost exclusively expressed in the brain and spinal cord compared to other tissues such as lung, liver and kidney. A similar pattern of central nervous system tissue expression of PAK-3 transcripts of 3.6 and 8kb was also observed. Analysis of two multilocus genetic crosses localized Pak1 and Pak3 to a position on chromosome 7 and X, respectively. The high level of PAK-1 and PAK-3 kinase expression in the mouse brain and spinal cord suggests a potentially important role for these kinases in the control of the cellular architecture and/or signaling in the central nervous system.

A limitation of current gene therapy efforts aimed at central nervous system disorders concerns distribution of vectors on direct injection into neural tissue. Here we have circumvented this problem by transferring genes to the meninges surrounding the spinal cord, achieving an in vivo gene transfer paradigm for treating chronic pain. The therapeutic vector consisted of a recombinant adenovirus encoding a secreted form of the potent endogenous opioid beta-endorphin. In an inflammation model of persistent pain, administration of the vector into the cerebrospinal fluid (CSF) surrounding the spinal cord transduced meningeal pia mater cells. The resulting increase in beta-endorphin secretion attenuated inflammatory hyperalgesia, yet had no effect on basal nociceptive responses. This demonstration of a gene transfer approach to pain treatment can be generalized to neurodegenerative disorders in which broad spatial distribution of therapeutic effect is critical.

The p190 family of GTPases consists of at least two different isoforms both containing an N-terminal GTPase and a C-terminal Rho GAP domain. Here we have isolated and characterized genomic and cDNA clones spanning the entire coding region of the mouse p190-B gene. Genomic data were obtained by sequencing plasmid subclones of two overlapping mouse genomic phage clones. Interestingly, a single 3.9 kb exon was found to contain approx. 80% of the coding region of the mouse p190-B protein (amino acid residues 1-1238) including the 5'-untranslated region, the N-terminal GTPase domain and a middle domain of unknown function. Missing from this exon, however, was the C-terminal Rho GAP domain, which was cloned from mouse brain mRNA using reverse transcriptase polymerase chain reaction. Comparison of the mouse with the human p190-B proteins revealed that approx. 97% of the amino acid residues were identical. Northern analysis of total RNA from a variety of mouse tissues detected ubiquitous expression of two p190-B transcripts of 4.0 and 6.8 kb in size. Analysis of two multilocus genetic crosses localized the mouse gene, Gfi2, to a position on chromosome 12, consistent with the mapping of the human gene to a position of conserved synteny on chromosome 14. The high level of sequence homology between the human and the mouse suggests that there is a strong selective pressure to maintain the p190-B protein structure.

Several lines of work demonstrate that there are two subtypes of kappa opioid receptors. Intrathecally administered agonists for the kappa1 subtype are not effective in treating pain, whereas agonists for the kappa2 receptor are anti-hyperalgesic and anti-allodynic. The question addressed here was whether the ratio of spinal kappa1 to kappa2 receptors was conserved across species. Thus, binding experiments were performed on spinal cord membranes from rats, guinea pigs, monkeys and humans. We found that kappa2 receptors were approximately ten times more abundant than kappa1 receptors in all species tested. This suggests that the anti-hyperalgesic and anti-allodynic properties of kappa2 agonists may also be conserved. Therefore, selective kappa2 agonists may be effective in treating chronic pain in humans.

A plasma membrane iron reductase, required for cellular iron acquisition by Saccharomyces cerevisiae, and the human phagocytic NADPH oxidase, implicated in cellular defense, contain low potential plasma membrane b cytochromes that share elements of structure and function. Four critical histidine residues in the FRE1 protein of the iron reductase were identified by site-directed mutagenesis. Individual mutation of each histidine to alanine eliminated the entire heme spectrum without affecting expression of the apoprotein, documenting the specificity of the requirement for the histidine residues. These critical residues are predicted to coordinate a bis-heme structure between transmembrane domains of the FRE1 protein. The histidine residues are conserved in the related gp91(phox) protein of the NADPH oxidase of human granulocytes, predicting the sites of heme coordination in that protein complex. Similarly spaced histidine residues have also been implicated in heme binding by organelle b cytochromes with little overall sequence similarity to the plasma membrane b cytochromes. This bis-heme motif may play a role in transmembrane electron transport by distinct families of polytopic b cytochromes.

Protein prenylation occurs by modification of proteins with one of at least two isoprenoids, the farnesyl group and the geranylgeranyl group. Protein farnesyltransferases have been identified, but no such enzyme has been identified for geranylgeranylation. We report the identification of an activity in crude soluble yeast extracts that catalyzes the transfer of a geranylgeranyl moiety from geranylgeranyl pyrophosphate to proteins having the C-terminal sequence Cys-Ile-Ile-Leu or Cys-Val-Leu-Leu but not to a similar protein ending with Cys-Ile-Ile-Ser. This activity is dependent upon the CDC43/CAL1 gene, which is involved in budding and the control of cell polarity, but does not require the DPR1/RAM1 gene, which is known to be required for the farnesylation of Ras proteins. These results indicate that the protein geranylgeranyltransferase activity is distinct from the protein farnesyltransferase activity and that its specificity depends in part on the extreme C-terminal leucine in the protein to be prenylated.

The heterotrimeric guanine nucleotide-binding regulatory proteins act at the inner surface of the plasma membrane to relay information from cell surface receptors to effectors inside the cell. These G proteins are not integral membrane proteins, yet are membrane associated. The processing and function of the gamma subunit of the yeast G protein involved in mating-pheromone signal transduction was found to be affected by the same mutations that block ras processing. The nature of these mutations implied that the gamma subunit was polyisoprenylated and that this modification was necessary for membrane association and biological activity. A microbial screen was developed for pharmacological agents that inhibit polyisoprenylation and that have potential application in cancer therapy.

Using the coil planet centrifugation method, the mechanism of hemolysis by alcohols and saponin was investigated. With this technique, erythrocytes are introduced into a gradient of hemolytic agents in saline, which is prepared in a long coiled polyethylene tube. The tube is centrifuged so that the cells move from a low to a high concentration of hemolytic agent. When the cells lyse, they release hemoglobin which remains stationary, and therefore hemolytic potency can be determined spectrophotometrically by the distance the cells move before lysing. We found that alcohols caused hemolysis at a particular concentration, whereas saponin-induced hemolysis was dependent on the amount of saponin accumulated in the environment of the cell. In addition, alcohols with longer carbon chains were more potent hemolytic agents than those with shorter chains, but each additional carbon group produced less of an increase in hemolysis per mole of alcohol. This chain-length dependency is consistent with a previous study on in vivo alcohol-induced hemolysis. The coil planet centrifugation method is also adaptable to comparative studies on the mechanism of other types of hemolysis, such as immune or drug-induced lysis, and to toxicological studies.