Some properties of an established fish cell line fromXiphophorus helleri (red swordtail)

Summary A cell line (SWT) was established from embryonic tissue of the red swordtailXiphophorus helleri. The SWT cells grew optimally, at 26°C to 30°C in Eagle's basal medium plus 10% fetal calf serum but failed to grow at 16°C and 37°C. After 50 subcultivations, the cells remained contact-inhibited and were pseudodiploid with a chromosomal modal number of 46. Virological studies demonstrated that SWT cells supported replication of the following viruses at the indicated temperatures: IPN virus (22°C), FV-3 (30°C), and VSV (33°C). The following mammalian, viruses failed to replicate at 33°C: vaccinia, poliovirus 2, herpes simplex, and reovirus 2. Although not replicating, reovirus induced interferon in there cells. This work was supported in part by a grant from the University Research Council. A part of these results was presented at the 22nd Annual Meeting of the Tissue Culture Association, Lake Placid, N. Y. 1971.     

The exchange of Fe3+ between pyrophosphate and transferrin. Probing the nature of an intermediate complex with stopped flow kinetics, rapid multimixing, and electron paramagnetic resonance spectroscopy

A detailed study of the exchange of Fe3+ between pyrophosphate and human serum transferrin was undertaken to test the hypothesis of a generalized reaction route for exchange of Fe3+ between transferrin and chelators. The initial rate of Fe3+ transfer from pyrophosphate to apotransferrin-CO2-3 is highly sensitive to the pyrophosphate to iron ratio with a maximal rate being observed at a ratio of 3:1, consistent with the presence of slowly reactive polymeric species at ratios less than 3:1 as revealed by EPR and kinetic measurements. At a ratio of 4:1 the reaction is distinctly biphasic. The rapid first phase results in the formation of an intermediate postulated as a mixedligand complex of the type PPi-Fe3+-transferrin-CO2-3. The intermediate has a distinct EPR spectrum and an absorption spectrum similar to that of Fe3+-transferrin-CO2-3, but with a spectral maximum at 450 nm rather than 465 nm. The second phase principally arises from the slow reaction of polymeric iron-pyrophosphate with the apoprotein and has contributions from the breakdown of the intermediate formed in the first phase. The rate of formation of the intermediate shows a hyperbolic dependence on NaHCO3 and apotransferrin concentrations, the latter suggesting a rate-limiting labilization of Fe3+(PPi)3, perhaps to form species of the type Fe3+(PPi)2, prior to attack by apotransferrin-CO2-3. Multimixing stopped flow spectrophotometry was employed to test the chemical reactivity of the Fe3+ to reduction at various times during the first phase. Surprisingly, a diminution of reactivity of 1000-fold was noted after only 2% of the first phase was completed, indicating a fast initial reaction which is not observed by normal rapid flow spectrophotometry. This initial reaction may involve the binding of iron-pyrophosphate to allosteric sites on the protein. The kinetics of iron removal from Fe3+-transferrin-CO2-3 by PPi are consistent with a rate-limiting conformational change in the protein as proposed earlier.     

A comparison of the major lipid classes and fatty acid composition of marine unicellular cyanobacteria with freshwater species

The fatty acid composition of two motile (strains WH 8113 and WH 8103) and one nonmotile (strain WH 7803) marine cyanobacteria has been determined and compared with two freshwater unicellular Synechocystis species (strain PCC 6308 and PCC 6803). The fatty acid composition of lipid extracts of isolated membranes from Synechocystis PCC 6803 was found to be identical to that of whole cells. All the marine strains contained myristic acid (14:0) as the major fatty acid, with only traces of polyunsaturated fatty acids. This composition is similar to Synechocystis PCC 6308. The major lipid classes of the nonmotile marine strain were identified as digalactosyl diacylglycerol, monogalactosyl diacylglycerol, phosphatidylglycerol, and sulfoquinovosyl diacylglycerol, identical to those found in other cyanobacteria.Abbreviations DGDG Digalactosyl diacylglycerol - MGDG Monogalactosyldiacylglycerol - PG Phosphatidylglycerol - SGDG sulfoquinovosyl diacylglycerol - gc gas chromatography - ms mass spectrometry     

T-cell receptor V Tβ genes in natural populations of mice

The composition of 15 V _T gene subfamilies has been examined by Southern hybridization among a broad spectrum of colony bred rat and mouse species extending phylogenetically from Rattus to Mus musculus domesticus. Most mouse species contain a similar content of V _T genes as determined by the number of hybridizing restriction fragment (RF) bands. Furthermore, the extent of restriction fragment length polymorphism (RFLP) appears to be limited. Some V _T gene families, however, are missing from Rattus (VT7, V _T12) and M. shortridgei (V _T9, V _T16). Extension of the V _T survey to a panel of 38 wild-caught mice reveals that nearly a third lack specific hybridization to the V _T5 probe. Previous reports have established that the mouse inbred strains SJL, C57BR, C57L, and SWR lack 50% of their V _T repertoire, including V _T5 (Behlke et al. 1985). This study demonstrates that natural populations of mice also carry a significantly reduced V _T gene repertoire.     

Molecular characterization of a new immunoglobulin superfamily protein with potential roles in opioid binding and cell contact.

A purified opioid-binding protein has been characterized by cDNA cloning. The cDNA sequence predicts an extracellularly located glycoprotein of 345 amino acids. This protein does not possess a membrane-spanning domain but contains a C-terminal hydrophobic sequence characteristic of membrane attachment by a phosphatidylinositol linkage. It displays homology to the immunoglobulin protein superfamily, featuring three domains that resemble disulfide-bonded constant regions. More specifically, the protein is most homologous to a subfamily of proteins which includes the neural cell adhesion molecule (NCAM) and myelin-associated glycoprotein (MAG) and one subgroup of the tyrosine kinase growth factor receptors comprising the platelet-derived growth factor receptor (PDGF R), the colony-stimulating factor 1 receptor (CSF-1 R) and the c-kit protooncogene. These sequence homologies suggest that the protein could be involved in either cell recognition and adhesion, peptidergic ligand binding or both.     

Pharmacological manipulation of gamma-aminobutyric acid (GABA) in morphine analgesia,tolerance and physical dependence

The findings from our laboratory indicated that pharmacological manipulations of GABA system modified morphine analgesia, tolerance and physical dependence. Elevating brain levels of GABA by slowing its destruction with aminooxyacetic acid not only antagonized the analgesic action of morphine in both non-tolerant and tolerant mice, but also enhanced the development of tolerance and physical dependence. On the other hand, blockade of postsynaptic sites of GABA receptors by bicuculline resulted in an inhibition of tolerance and dependence development. Administration of 2,4-diaminobutyric acid, an inhibitor of GABA uptake in the neurons, antagonized morphine analgesia in both non-tolerant and tolerant mice. However, it did not modify naloxone precipitated withdrawal jumping. On the contrary, β-alanine, an inhibitor of the GABA uptake process in glial cells, potentiated naloxone precipitated withdrawal jumping in morphine dependent mice, but it had no effect on morphine antinociception in both non-tolerant and tolerant mice.     

Distinct Differences Between Morphine- and [d-Ala2,N-MePhe4,Gly-ol5]-Enkephalin- μ-Opioid Receptor Complexes Demonstrated by Cyclic AMP-Dependent Protein Kinase Phosphorylation

Abstract: The present study demonstrates a conditional, agonist-dependent phosphorylation of the μ-opioid receptor (MOR-1) by cyclic AMP-dependent protein kinase (PKA) in membrane preparations of MOR-1-transfected neuroblastoma Neuro2A cells. Opioid agonist-dependent phosphorylation occurs in a time- and concentration-dependent manner (EC₅₀∼40 n M ) and can be abolished by the receptor antagonist naloxone. Stoichiometric analysis indicates incorporation of a maximum of 6 mol of phosphate/mol of receptor in the presence of 1 µ M morphine and 6 n M PKA. Although morphine and related alkaloids as well as some peptide agonists (PLO17 and β-endorphin) stimulated phosphorylation of MOR-1 by PKA, the potent μ-opioid-selective peptide [ d -Ala², N -MePhe⁴,Gly-ol⁵]-enkephalin (DAMGO) or other enkephalin analogues such as [ d -Ala²]-Met⁵-enkephalinamide (DALA), [ d -Ala², d -Leu⁵]-enkephalin (DADLE), and Met⁵-enkephalin had no effect. The lack of the effect of DAMGO on MOR-1 phosphorylation state was evident also after chronic pretreatment. These results suggest the existence of different agonist-dependent conformations of MOR-1. Furthermore, phosphorylation may be a useful parameter with which to identify different agonist-receptor conformations.     

Comparisons of myeloma proteins from NZB and BALB/c mice: structural and functional differences of heavy chains.

The N-terminal sequences from heavy variable regions of 47 myeloma proteins of the NZB mouse have been analyzed. Sixteen of these VH regions have unblocked alpha amino groups and have been analyzed over their N-terminal 20 residues by automatic sequence analysis. These sequence data along with the antigen-binding profiles and immunoglobulin class distribution are compared with comparable data from BALB/c myeloma proteins. These comparisons suggest that the NZB and BALB/c populations of myeloma proteins are distinct from one another. The genetic implications of this conclusion are discussed.