α1-Adrenergic Receptor Mediates Arachidonic Acid Release in Spinal Cord Neurons Independent of Inositol Phospholipid Turnover

The alpha 1-adrenergic receptor has been shown to mediate the release of arachidonic acid in FRTL5 thyroid cells and MDCK kidney cells. In primary cultures of spinal cord cells, norepinephrine stimulated release of arachidonic acid (from neurons only) and turnover of inositol phospholipids (from neurons and glia) via alpha 1-adrenergic receptors. These two responses were dissociated by treatment with phorbol ester and pertussis toxin, which inhibited production of inositol phosphates with no appreciable effect on release of arachidonic acid. Extracellular calcium was required for release of arachidonic acid, but not for production of inositol phosphates. The calcium channel blockers nifedipine and verapamil inhibited release of arachidonic acid only. However, 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), a compound that blocks intracellular calcium release, diminished production of inositol phosphates, but had little effect on release of arachidonic acid. These results suggest that alpha 1-adrenergic receptors couple to release of arachidonic acid in primary cultures of spinal cord cells by a mechanism independent of activation of phospholipase C, possibly via the activation of phospholipase A2.     

DNA sequence of a class I pseudogene from theTla region of the murine MHC: Recombination at a B2 Alu repetitive sequence

Summary We have determined the DNA sequence of a BALB/cTla region class I gene from the major histocompatibility complex (MHC) that had been identified previously as encoding a murine antigen by DNA-mediated gene transfer. Analysis of the DNA sequence shows, however, that this gene, the T1^c gene from theTla ^c genotype, could not encode a TL antigen or any other functional class I molecule due to the presence of numerous stop codons and frameshift mutations in the coding regions. This result suggests that the earlier transformation data may have been incorrect or perhaps that the clone containing the T1^c gene contains sequences that induced expression of a serologically reactiveTla gene in the genome of the recipient L cell. The T1^c gene is structurally related to the previously sequenced T13^c gene that encodes a serologically defined TL antigen. The 3 half of the T1^c gene including exons 4, 5, 6, and the 3 untranslated region has about 85% nucleotide similarity (including introns) with the corresponding parts of the T13^c gene; however, the 5 half of the T1^c gene has little homology with the T13^c gene. There is a sharp line of demarcation between the homologous and nonhomologous regions, and this border occurs precisely at a B2 Alu repeat sequence present in the T13^c gene. This suggests that a recombination event took place here and that an Alu repeat sequence that is known to have characteristics of transposable elements played some role in a recombination or gene conversion event.     

The role of excision repair in the removal of transient benzo[a]pyrene-induced DNA lesions in Chinese hamster ovary cells

In Chinese hamster ovary (CHO) cells, benzo[a]pyrene induces both persistent and transient lesions that are detected by alkaline sucrose gradient sedimentation analysis (ASG sites). The transient lesions disappear within 15 min while the persistent lesions can be detected for several hours following treatment. Although the persistent ASG sites are believed to be repaired by excision repair, the process responsible for the disappearance of the transient ASG sites is unknown. To determine the contribution of excision repair to the removal of these transient lesions, CHO cells were treated with benzo[a]pyrene (B(a)P) in the presence of the inhibitors of excision repair, araC and novobiocin. The results indicate that: (1) araC inhibits the removal of persistent, but not the transient B(a)P-induced ASG sites; (2) novobiocin, a putative inhibitor of the incision step of DNA excision repair, reduced the number of lesions detected immediately following treatment, indicating that many of these lesions may represent single-strand discontinuities generated during repair; and (3) the lesions detected in the presence of novobiocin disappear rapidly following treatment. Based on these results, we concluded that B(a)P-induced transient ASG sites are repaired by a process other than excision repair.     

Analysis ofl-alanine:2-oxoglutarate aminotransferase isozymes in maize

Isozyme analysis ofl-alanine:2-oxoglutarate aminotransferase (ALT) in maize indicates that there are three genes encoding this enzyme activity. Two of the gene products interact with each other to form heterodimers, while the third gene product does not interact with the other two. Another isozyme that appears after gel electrophoresis and ALT staining is shown to be glutamate dehydrogenase-1. Anaerobic treatment does not result in increased ALT levels, indicating that the previously reported increase in alanine levels caused by this treatment may be due to increases in the level of pyruvate, a substrate of ALT.D. A. Russell was partially supported by a graduate student fellowship from the Division of Biology and Biomedical Sciences, Washington University. V. M. Peschke was partially supported by a postdoctoral fellowship from Monsanto. This research was supported by NIH Grant R01 GM34740.     

Two-step purification of full-length nerve growth factor receptor and maintenance of receptor-specific binding activity

A method for the purification of full-length nerve growth factor receptor (NGFRc) using membranes from three different cell lines was developed. We emphasized recovery of NGFRc that retained specific binding activity. Lipids were required to preserve binding activity during solubilization and throughout the purification procedure. Phosphatidylcholine was used for this purpose. Lectin affinity chromatography followed by high-resolution anion-exchange chromatography was used, and a 3000-fold increase in specific binding activity was obtained for NGFRc from human melanoma A875 membranes. Seven percent of the original binding activity was recovered as pure NGFRc. NGFRc binding activity eluted at 0.35 M NaCl in anion-exchange chromatography of solubilized A875, rat pheochromocytoma PC12, and human neuroblastoma MC-IXC membranes. Eight and three percent of the original binding activity were recovered as highly enriched NGFRc from membranes prepared from PC12 and MC-IXC cells, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of highly enriched, 125I-labeled NGFRc revealed several protein species. After chromatography, identification of proteins as NGFRc was verified both by immunoprecipitation using receptor-specific monoclonal antibodies and by covalent cross-linking to 125I-NGF using N-hydroxysuccinimidyl-4-azidobenzoate. Predominantly, NGFRc was recovered as a mixture of species of 80 and 160-180 kDa. Small amounts of larger species as well as smaller species were observed, consistent with minor amounts of receptor aggregation and proteolysis occurring during purification.     

Genetic variation of recent Alu insertions in human populations

The Alu family of intersperesed repeats is comprised of ovr 500,000 members which may be divided into discrete subfamilies based upon mutations held in common between members. Distinct subfamilies of Alu sequences have amplified within the human genome in recent evolutionary history. Several individual Alu family members have amplified so recently in human evolution that they are variable as to presence and absence at specific loci within different human populations. Here, we report on the distribution of six polymorphic Alu insetions in a survey of 563 individuals from 14 human population groups across several continents. Our results indicate that these polymorphic Alu insertions probably have an African origin and that there is a much smaller amount of genetic variation between European populations than that found between other populations groups. Present address: Department of Pathology, Stanley S. Scott Cancer Center, Louisiana State University Medical Center, 1901 Perdido St., New Orleans, LA 70112 Correspondence to: M.A. Batzer     

NMR Studies of Pi-Containing Extracellular and Cytoplasmic Compartments in Brain

Abstract: The inorganic phosphate (P_i) NMR peak in brain has an irregular shape, which suggests that it represents more than a single homogeneous pool of P_i. To test the ability of the Marquardt-Levenberg (M-L) nonlinear curve fit algorithm software (Peak-Fit) to separate multiple peaks, locate peak centers, and estimate peak heights, we studied simulated P_i spectra with defined peak centers, areas, and signal-to-noise (S/N) ratios ranging from ∞ to 5.8. As the S/N ratio decreased below 15, the M-L algorithm located peak centers accurately when they were detected; however, small peaks tended to grow smaller and disappear, whereas the amplitudes of larger peaks increased. We developed an in vitro three-compartment model containing a mixture of P_i buffer, phosphocreatine, phosphate diester, and phosphate monoester (PME), portions of which were adjusted to three different pHs before addition of agar. Weighed samples of each buffered gel together with phospholipid extract and bone chips were placed in an NMR tube and covered with mineral oil. Following baseline correction, it was possible to separate the P_i peaks arising from the three compartments with different pH values if each peak made up 10–35% of total P_i area. In vivo, we identified the plasma compartment by intraarterial infusion of P_i. It was assumed that intracellular compartments contained high-energy phosphates and took up glucose. Based on these assumptions we subjected the brains to complete ischemia and observed that P_i compartments at pH 6.82, 6.92, 7.03, and 7.13 increased markedly in amplitude. If the brain cells took up and phosphorylated 2-deoxyglucose (2-DG), 2-DG-6-phosphate (2-DG-6-P) would appear in the PME portion of the spectrum ionized according to pH_i. Four 2-DG-6-P peaks with calculated pH values of 6.86, 6.94, 7.04, and 7.15 did appear in the spectrum, thereby confirming that the four larger P_i peaks represented intracellular spaces.