Neurotensin interacts with dopaminergic neurons in rat brain

Neurotensin (NT) injected intracerebroventricularly in rat increases dopamine (DA) turnover in the corpus striatum and nucleus accumbens. Significant increases in 3,4-dihydroxyphenylacetic acid (DOPAC) levels occurred within 15 minutes after injection with peak levels at 60 minutes. The effect on NT on DOPAC and homovanillic acid (HVA) accumulation was dose-dependent at 3–100 μg. NT, like haloperidol, stimulated 3,4-dihydroxyphenylalanine (DOPA) accumulation in striatal neurons, in the presence of DOPA decarboxylase inhibitor, after injection of gamma-butyrolactone (GBL). NT had a similar stimulatory effect on DOPA levels in the accumbens while haloperidol (0.25 mg·kg^?1) had no significant effect in this brain region. NT did not block the inhibitory effect of apomorphine on DOPA accumulation in both the striatum and accumbens, while haloperidol inhibited apomorphine effect in both regions. NT also failed to displace ³H-spiperone from DA receptors and the presence of NT in the binding assay did not alter the ability of DA to displace ³H-spiperone in either brain region. These experiments demonstrate that NT increases DA turnover in both the nigrostriatal and mesolimbic pathways.     

Characterization of Lrp, and Escherichia coli regulatory protein that mediates a global response to leucine

Exogenous leucine affects the expression of a number of different operons in Escherichia coli. For at least some of these operons, the leucine-related effect is mediated by a protein called Lrp (Leucine-responsive regulatory protein). The purification of Lrp to near homogeneity is described. Lrp is a moderately abundant, basic protein composed of two subunits of molecular mass 18.8 kDa each. In addition, the corresponding protein was purified from a strain having a mutation within the gene that encodes Lrp (lrp). This mutation (lrp-1) causes high constitutive expression of ilvIH, one of the operons controlled by Lrp (Platko, J. V., Willins, D.A., and Calvo, J.M. (1990) J. Bacteriol. 172, 4563-4570). The Lrp-1 and Lrp proteins have similar physical properties, but they show some differences in the characteristics with which they bind DNA upstream of the ilvIH promoter. The nucleotide sequences of the lrp and lrp-1 genes differ by only a single nucleotide, a C to G change that would substitute a Glu for an Asp at amino acid 114. Lrp has some amino acid sequence similarity to AsnC, a protein that regulates asnA expression (Kolling, R., and Lother, H. (1985) J. Bacteriol. 164, 310-315).     

Residues in the alpha subunit of human choriotropin that are important for interaction with the lutropin receptor

Synthetic peptides were used to probe the structure-function relationships between human choriotropin (hCG) and the lutropin (LH) receptor. Previously, a peptide region of the alpha subunit of hCG, residues 26-46, had been shown to inhibit binding of 125I-hCG to the LH receptor in rat ovarian membranes (Charlesworth, M.C., McCormick, D.J., Madden, B., and Ryan, R.J. (1987) J. Biol. Chem. 262, 13409-13416). To determine which residues are important for this inhibitory activity, peptides were truncated from either the amino or carboxyl terminus, or individual residues were substituted with alanine. The amino-terminal boundary was determined to be Gly-30 and the carboxyl-terminal boundary, Lys-44. This core peptide contained all the residues needed for full activity of the parent peptide 26-46. Arg-35 and Phe-33 were particularly important residues; when they were substituted with alanine, the peptide inhibitory potencies were decreased. Ser-43, Arg-42, Cys-32, and Cys-31 were also important but to a lesser degree. These results are consistent with predictions based on chemical and enzymatic modification studies and provide insight into which residues are important for interaction between hCG and the LH receptor.     

NKR-P1, an activating molecule on rat natural killer cells, stimulates phosphoinositide turnover and a rise in intracellular calcium

NKR-P1 is a 60-kDa homodimer expressed on all rat NK cells. Previous studies by others suggest that NKR-P1 may play a role in NK cell activation because antibody to NKR-P1 stimulates the release of granules from NK cells, and anti-NKR-P1 causes redirected lysis by activated NK cells against targets that express FcR. To examine the mechanism of transmembrane signaling by NKR-P1, we studied the rat NK cell line, RNK-16. We here demonstrate that F(ab')2 antibody to NKR-P1 stimulates phosphoinositide turnover and a rise in intracellular calcium within RNK-16 cells. The response is augmented by cross-linking the F(ab')2 antibody. The phosphoinositide/calcium pathway is also stimulated by NKR-P1 in activated rat NK cells, although no response is detectable in polymorphonuclear cells, which also express NKR-P1. We also demonstrate that RNK-16 cells kill the anti-NKR-P1 (3.2.3) hybridoma and that exposure to the hybridoma target cells stimulates phosphoinositide turnover in RNK-16 cells. Both killing and phosphoinositide turnover are inhibited by F(ab')2 anti-NKR-P1, implicating NKR-P1 in both responses. In contrast, neither cytotoxicity nor phosphoinositide turnover is appreciably blocked by F(ab')2 anti-NKR-P1 in response to YAC-1 targets. Thus, with either target, killing is linked to phosphoinositide turnover, but killing of YAC-1 involves pathways that differ from those that direct killing of the anti-NKR-P1 hybridoma. Our studies support the hypothesis that NKR-P1 may serve as an activating cell-surface receptor on NK cells, and they clarify the mechanisms by which it activates NK cells.     

Analysis of Actinobacillus pleuropneumoniae and related organisms by DNA-DNA hybridization and restriction endonuclease fingerprinting.

The objective of this study was to determine the degree of genetic relatedness of Actinobacillus pleuropneumoniae to selected members of the family Pasteurellaceae, with particular emphasis on species commonly associated with swine. Free-solution DNA-DNA hybridization studies revealed that representative strains of all 12 serotypes of A. pleuropneumoniae formed a homogeneous group, sharing 74 to 90% sequence homology with A. pleuropneumoniae serotype 1. All serotypes of A. pleuropneumoniae tested demonstrated a high degree of genetic relatedness (66 to 79%) to the type species of the genus Actinobacillus, A. lignieresii. Little homology (less than 20%) was detected between A. pleuropneumoniae strains and selected Haemophilus spp. and Pasteurella spp. Since free-solution hybridization methods are technically demanding and require large amounts of highly purified DNA, restriction endonuclease fingerprinting (REF) was examined to determine whether it could be a useful taxonomic tool for classification of members of the family Pasteurellaceae. REF profiles were compared, and the degree of similarity between organisms was quantitated by calculating Jaccard similarity coefficients. There was a significant positive relationship between the REF Jaccard coefficients and the DNA homology values determined from free-solution hybridization experiments.     

Morphology and electrical properties of Schwann cells around the giant axon of the squids Loligo forbesi and Loligo vulgaris.

The first successful dye-fills of Schwann cells around the split giant axon of Loligo show them to be spindle-shaped cells ca. 600 microns long and 20 microns wide lying parallel to the axonal axis. There are some 50,000 Schwann cells per cm2 of axonal membrane. Only a small part (ca. 6% of each Schwann cell membrane) is in contact with the periaxonal space, the remainder is overlain by adjacent Schwann cells, or applied to the basal lamina. The mean membrane potential of the Schwann cells in artificial seawater (ASW) varies from around -40 mV in fresh split-axon preparations to around -60 to -70 mV after 1-2 h; this hyperpolarization is not seen in preparations dissected and maintained in Ca2(+)-free ASW. Electrical- and dye-coupling (abolished by prior octanol treatment) is present between Schwann cells, but is weaker in cells with lower (less negative) membrane potentials. The implications for potassium homeostasis around the axon are briefly discussed.     

Structural implications of spectroscopic characterization of a putative zinc finger peptide from HIV-1 integrase.

The N-terminal domain of human immunodeficiency virus (HIV-1) integrase (IN) contains the sequence motif His-Xaa3-His-Xaa23-Cys-Xaa2-Cys, which is strongly conserved in all retroviral and retrotransposon IN proteins. This structural motif constitutes a putative zinc finger in which a metal ion may be coordinately bound by the His and Cys residues. A recombinant peptide, IN(1-55), composed of the N-terminal 55 amino acids of HIV-1 IN was expressed in Escherichia coli and purified. Utilizing a combination of techniques including UV-visible absorption, circular dichroism, Fourier transform infrared, and fluorescence spectroscopies, we have demonstrated that metal ions (Zn2+, Co2+, and Cd2+) are bound with equimolar stoichiometry by IN(1-55). The liganded peptide assumes a highly ordered structure with increased alpha-helical content and exhibits remarkable thermal stability. UV-visible difference spectra of the peptide-Co2+ complexes directly implicate thiols in metal coordination, and Co2+ d-d transitions in the visible range indicate that Co2+ is tetrahedrally coordinated. Mutant peptides containing conservative substitutions of one of the conserved His or either of the Cys residues displayed no significant Zn(2+)-induced conformational changes as monitored by CD and fluorescence spectra. We conclude that the N terminus of HIV-1 IN contains a metal-binding domain whose structure is stabilized by tetrahedral coordination of metal by histidines 12 and 16 and cysteines 40 and 43. A preliminary structural model for this zinc finger is presented.     

Conversion of human low density lipoprotein into a very low density lipoprotein-like particle in vitro.

The lipid substrate specificity of Manduca sexta lipid transfer particle (LTP) was examined in in vitro lipid transfer assays employing high density lipophorin and human low density lipoprotein (LDL) as donor/acceptor substrates. Unesterified cholesterol was found to exchange spontaneously between these substrate lipoproteins, and the extent of transfer/exchange was not affected by LTP. By contrast, transfer of labeled phosphatidylcholine and cholesteryl ester was dependent on LTP in a concentration-dependent manner. Facilitated phosphatidylcholine transfer occurred at a faster rate than facilitated cholesteryl ester transfer; this observation suggests that either LTP may have an inherent preference for polar lipids or the accessibility of specific lipids in the donor substrate particle influences their rate of transfer. The capacity of LDL to accept exogenous lipid from lipophorin was investigated by increasing the high density lipophorin:LDL ratio in transfer assays. At a 3:1 (protein) ratio in the presence of LTP, LDL became turbid (and aggregated LDL were observed by electron microscopy) indicating LDL has a finite capacity to accept exogenous lipid while maintaining an overall stable structure. When either isolated human non B very low density lipoprotein (VLDL) apoproteins or insect apolipophorin III (apoLp-III) were included in transfer experiments, the sample did not become turbid although lipid transfer proceeded to the same extent as in the absence of added apolipoprotein. The reduction in sample turbidity caused by exogenous apolipoprotein occurred in a concentration-dependent manner, suggesting that these proteins associate with the surface of LDL and stabilize the increment of lipid/water interface created by LTP-mediated net lipid transfer. The association of apolipoprotein with the surface of modified LDL was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, and scanning densitometry revealed that apoLp-III bound to the surface of LDL in a 1:14 apoB:apoLp-III molar ratio. Electron microscopy showed that apoLp-III-stabilized modified LDL particles have a larger diameter (29.2 +/- 2.6 nm) than that of control LDL (22.7 +/- 1.9 nm), consistent with the observed changes in particle density, lipid, and apolipoprotein content. Thus LTP-catalyzed vectorial lipid transfer can be used to introduce significant modifications into isolated LDL particles and provides a novel mechanism whereby VLDL-LDL interrelationships can be studied.     

Inhibition of human bone marrow lymphoid progenitor colonies by antibodies to VLA integrins.

Studies in animal models suggest that the integrin adhesion protein VLA-4 may play an important role in lymphopoiesis. The relationship between cell adhesion and lymphopoiesis in humans has been difficult to study because of the relative rarity and stringent in vitro growth requirements of lymphoid progenitors from normal adult human bone marrow. To determine the functional significance of VLA-4-mediated adhesion in human lymphopoiesis, we developed a culture system in which a bone marrow-derived adherent layer supports the formation of colonies of terminal deoxynucleotidyl transferase (TdT)-positive lymphoid precursor cells from normal adult human bone marrow. Limiting dilution studies were consistent with clonal origin of these colonies. CFU-TdT were enriched in the CD34+ bone marrow fraction, consistent with CD34 expression by other hematopoietic progenitors. CD34 expression and lack of lineage-specific markers in a significant proportion of the TdT+ colony cells suggest that the TdT+ CFU may represent an uncommitted lymphoid progenitor cell. Development of TdT+ colonies required direct contact with the adherent layer and was significantly inhibited by specific anti-VLA-4 alpha chain antibody, suggesting a functional role for the previously reported VLA-4-dependent adhesion of human B cell precursors to bone marrow-derived fibroblasts.     

Factors affecting chloride conductance in apical membrane vesicles from human placenta

Summary Apical membrane vesicles from human term placenta were isolated using a magnesium precipitation technique, and the purity of the vesicles was assessed morphologically using scanning and transmission electron microscopy, and biochemically, using marker enzymes. The vesicles were found to be morphologically intact and significantly enriched in enzymes associated with apical membranes. ³⁶Cl^– uptake into these vesicles was studied in the presence of an outwardly directed Cl^– gradient. This uptake was found to be time dependent, with an initial rapid uptake tending to peak between 10 and 20 min and thereafter decline. Uptake was found to be voltage dependent since 5 m valinomycin caused a decrease in uptake. The effects of N-phenylanthranilic acid (NPA) and 4,4-diisothiocyanostilbene-2,2-disulphonic acid (DIDS) and bumetanide on the initial rate of Cl^– were examined in the presence and absence of 5 m valinomycin. NPA and DIDS inhibited isotope uptake strongly with IC₅₀ values of 0.83±0.35 m and 3.43±0.37 m, respectively, in the absence of valinomycin. Although valinomycin reduced ³⁶Cl^– uptake by about 80% when added before the isotope, DIDS reduced the uptake which remained in a concentration-dependent fashion with an IC₅₀ of 5.6±2.1 m. Under these conditions, NPA was without effect at concentrations below 100 m. Bumetanide was without effect at the concentrations used in the absence of valinomycin. However, following valinomycin pretreatment, bumetanide reduced ³⁶Cl^– uptake significantly at 100 m concentration. Vesicle diameter, as assessed by flow cytometry, did not change under the conditions employed.The effects of some fatty acids were also investigated. Arachidonic acid and linoleic acid inhibited Cl^– uptake with IC₅₀ values of 37.6±14.9 m and 4.59±0.51 m, respectively. Arachidonyl alcohol and elaidic acid were found to be without effect. These studies show that human placental brush border membrane vesicles possess a chloride conductance channel, the activity of which can be measured in the presence of an outwardly directed Cl^– gradient and this channel is sensitive to Cl^– channel inhibitors, especially N-phenylanthranilic acid, and can be inhibited by unsaturated fatty acids such as arachidonic acid and linoleic acid.This work was supported in part by the Cystic Fibrosis Association of Ireland and Eolas, The Irish Science and Technology Agency. The technical assistance of Mr. Cormac O' Connell in the preparation of the electron micrographs and of Mr. Roddy Monks in the flow cytometric analysis is gratefully acknowledged.