Identification and Function of Glycine Receptors in Cultured Cerebellar Granule Cells

Abstract: Poly(A)⁺ mRNA was isolated from cultured mouse cerebellar granule cells and injected into Xenopus oocytes. This led to the expression of receptors that evoked large membrane currents in response to glycine. Current-responses were also obtained after application of β-alanine and taurine, but these were very low relative to that of glycine (maximal β-alanine and taurine responses were 8 and 3% of that of glycine, respectively). The role of glycine receptors on K⁺-evoked transmitter release in cultured cerebellar granule cells was also assayed. Release of preloaded d -[³H]aspartate evoked by 40 m M K⁺ was dose dependently inhibited by glycine, and the concentration producing half-maximal inhibition was 50 μ M. Taurine, β-alanine, and the specific GABA_A receptor agonist isoguvacine also inhibited K⁺-evoked release, and the maximal inhibition was similar for all agonists (˜40%). The EC₅₀ value was 200 μ M for taurine, 70 μ M for β-alanine, and 4 μ M for isoguvacine. Bicuculline (150 μ M ) antagonized the inhibitory effect of isoguvacine (150 μ M ) but not that of glycine (1 m M ). In contrast, strychnine (20 μ M ) antagonized the inhibitory effect of glycine (1 m M ) but not that of isoguvacine (150 μ M ). The pharmacology of the responses to β-alanine and taurine showed that these agonists activate both glycine and GABA_A receptors. The results indicate that cultured cerebellar granule cells translate the gene for the glycine receptor and that activation of glycine receptors produces neuronal inhibition.     

Thapsigargin-induced calcium entry in FRTL-5 cells: Possible dependence on phospholipase A2 activation

Stimulating rat thyroid FRTL-5 cells with agonists that activate the inositol phosphate cascade results in the release of sequestered calcium and influx of extracellular calcium. In addition, phospholipase A₂ (PLA₂) is activated. Since PLA₂ is a calcium-dependent enzyme we wanted to investigate the interrelationships between PLA₂ activity and the entry of calcium. Stimulating ³H-arachidonic acid (³H-AA)-labelled cells with thapsigargin resulted in a substantial release of ³H-AA. This release was totally abolished in a calcium-free buffer. Pretreatment of Fura 2 loaded cells with 4-bromophenacyl bromide, an inhibitor of PLA₂ activity, decreased the thapsigargin-induced entry of calcium, suggesting a role for PLA₂ in the regulation of calcium entry. In cells treated with nordihydroguaiaretic acid (NDGA), clotramizole, or econazole, compounds with lipoxygenase and cytochrome P-450 inhibitory actions, the thapsigargin-induced entry of calcium was decreased in a dose-dependent manner. However, treatment of the cells with indomethacin, a cyclooxygenase inhibitor, had no effect on the thapsigargin-induced calcium entry. We also showed that stimulation of the cells with arachidonic acid released sequestered calcium, apparently from the same intracellular pool as did thapsigargin. The results suggested that the calcium-induced PLA₂ activation and the metabolism of the produced arachidonic acid by a noncyclooxygenase pathway may be of importance in maintaining calcium entry after releasing sequestered Ca²⁺ in FRTL-5 cells. © 1994 Wiley-Liss, Inc.     

Effects of seawater-acclimatization and release sites on survival of hatchery–reared brown trout Salmo trutta

To test whether seawater–acclimatization of hatchery–reared anadromous and freshwater resident brown trout before release increased the survival of adults, smolts were retained 0, 2, 4 and 8 weeks in sea water before release. Total recapture rate increased for smolts retained 4 and 8 weeks in sea water before release relative to the controls. This trend was more pronounced for Freshwater resident than for anadromous stocks, Offspring of anadromous fish stayed longer at sea than offspring of freshwater resident fish. Recapture rates in fresh water were higher for brown trout released in the river than in the fjord in the R, Drammen area, but not in the R. Imsa. In both cases, most fish were recaptured in the sea. Moving into the R. Imsa (relative to other rivers) appeared higher for fish released at the mouth of the river (93%) than in the fjord (47%). Judged from the recapture rates, sea survival appeared to be the same whether released in the fjord or at the mouth of the river.     

Differential response to water current in offspring of inlet-and outlet-spawning brown trout Salmo trutta

Two-year-old hatchery-reared progeny of inlet- and outlet spawning brown trout from Lake Tytifjorden were released at the mouth of the R. Imsa, south-western Norway. There were significant differences in migratory direction of juveniles between the two populations. After release, juvenile fish from the outlet river population moved against the current and ascended the R Imsa, while the inlet rivet fish tended to migrate with the water current to the sea. This differential response to water current in juveniles appears to be due to genetic differences between the populations, and parallels that found in their ancestors native environments.     

Functional domains of Moloney murine leukemia virus integrase defined by mutation and complementation analysis.

Retroviral integrases perform two catalytic steps, 3' processing and strand transfer, that result in the stable insertion of the retroviral DNA into the host genome. Mutant M-MuLV integrases were constructed to define the functional domains important for 3' processing, strand transfer, and disintegration by in vitro assays. N-terminal mutants had no detectable 3' processing activity, and only one mutant which lacks the HHCC domain, Ndelta105, had strand transfer activity. Strand transfer mediated by Ndelta105 showed preference for one site in the target DNA. Disintegration activity of N-terminal mutants decreased only minimally. In contrast, all C-terminal mutants truncated by more than 28 amino acids had no integration or disintegration activity. Activity on a single-strand disintegration substrate did not require a functional HHCC domain but did require most of the C-terminal region. Complementation analysis found that the HHCC region alone was able to function in trans to a promoter containing only the DD(35)E and C-terminal regions and to enhance integration site selection. Increasing the reducing conditions or adding the HHCC domain to Ndelta105 reaction mixtures restored the wild-type strand transfer activity and range of target sites. The reducing agent affected Cys-209 in the DD(35)E region. The presence of C-209 was required for complementation of Ndelta105 by the HHCC region.     

Coordinated disintegration reactions mediated by Moloney murine leukemia virus integrase.

The protein-DNA and protein-protein interactions important for function of the integrase (IN) protein of Moloney murine leukemia virus (M-MuLV) were investigated by using a coordinated-disintegration assay. A panel of M-MuLV IN mutants and substrate alterations highlighted distinctions between the intermolecular and intramolecular reactions of coordinated disintegration. Mispairing of the crossbone single-strand region and altered long terminal repeat (LTR) positioning affected the intermolecular, but not the intramolecular, reactions of coordinated disintegration. Partial components of the crossbone substrate were coordinated by M-MuLV IN, indicating a reliance on both LTR and target DNA determinants for substrate assembly. The intramolecular reaction was dependent on the presence of either the HHCC domain or a crossbone LTR 5' single-stranded tail. An M-MuLV IN mutant without the HHCC domain (Ndelta105) catalyzed reduced levels of double disintegration but not single disintegration. A separately purified HHCC domain protein (Cdelta232) stimulated double disintegration mediated by Ndelta105, suggesting a role of the N-terminal HHCC domain in stable IN-IN and IN-DNA interactions. Significantly, crossbone substrates lacking the LTR 5' tails were not recognized by the fingerless Ndelta105 protein. Collectively, these data suggest similar roles of the HHCC domain and 5' LTR tail in substrate recognition and modulation of IN activity.     

Molecular characterization of patients with 18q23 deletions.

The 18q- syndrome is a deletion syndrome that is characterized by mental retardation, hearing loss, midfacial hypoplasia, growth deficiency, and limb anomalies. Most patients with this syndrome have deletions from 18q21-qter. We report on three patients with deletions of 18q23. A mother and daughter with identical deletions of 18q23 have many of the typical features of the 18q- syndrome, including midfacial hypoplasia and hearing loss. In contrast, the third patient has few of the symptoms of the 18q- syndrome. A contig of the 18q23 region was generated to aid in the mapping of the breakpoints. FISH was used to map both breakpoints to the same YAC clone. Furthermore, somatic-cell hybrids from the daughter and the third patient were isolated. The mapping results of sequence-tagged sites relative to the two breakpoints were identical, suggesting that the two deletion breakpoints map very close to one another. The analyses of these patients demonstrate that the critical region for the 18q- syndrome maps to 18q23 but that a deletion of 18q23 does not always lead to the clinical features associated with the syndrome. These patients demonstrate the wide phenotypic variability associated with deletions of 18q.     

An imidazole based H-Phe-Phe-NH2 peptidomimetic with anti-allodynic effect in spared nerve injury mice

The dipeptide amide H-Phe-Phe-NH₂ (1) that previously was identified as a ligand for the substance P 1–7 (SP_1–7) binding site exerts intriguing results in animal models of neuropathic pain after central but not after peripheral administration. The dipeptide 1 is derived from stepwise modifications of the anti-nociceptive heptapeptide SP_1–7 and the tetrapeptide endomorphin-2 that is also binding to the SP_1–7 site. We herein report a strong anti-allodynic effect of a new H-Phe-Phe-NH₂ peptidomimetic (4) comprising an imidazole ring as a bioisosteric element, in the spare nerve injury (SNI) mice model after peripheral administration. Peptidomimetic 4 was stable in plasma, displayed a fair membrane permeability and a favorable neurotoxic profile. Moreover, the effective dose (ED₅₀) of 4 was superior as compared to gabapentin and morphine that are used in clinic.     

Rapid, high-throughput purification of HIV-1 integrase using microtiter plate technology

The human immunodeficiency virus type-1 (HIV-1) integrase (IN) catalyzes the insertion of the retroviral genome into the chromosome of an infected host cell. HIV-1 IN was expressed as a N-terminal hexa-histidine fusion in Escherichia coli. A high-throughput purification strategy was developed using denaturing methods for the initial protein extraction, followed by a one-step nickel-chelating chromatography purification and step-wise refolding. IN was routinely greater than 90% pure with yields exceeding 14 microg of purified IN per ml of E. coli culture. In vitro 3' processing and strand transfer assays showed the enzyme preparations to be highly active. The specific activity of the purified IN was 2.65 pmol/h/microg IN, which is very similar to the activity of IN routinely produced by large-scale column chromatographic methods. This high-throughput platform should be of general utility to those interested in defining the structure-function relationship of proteins and enzymes.