Potassium channel blockers inhibit adoptive transfer of experimental allergic encephalomyelitis by myelin-basic-protein-stimulated rat T lymphocytes

Agents which block T cell K⁺ currents can prohibit both proliferative and effector cell functions in T cells activated by mitogens or phorbol esters. This study examined the effects of some of these blocking agents on the immune responsiveness of guinea pig myelin basic protein (GPMBP)-reactive Lewis rat T lymphocytes, which are capable of mediating the adoptive transfer of experimental allergic encephalomyelitis (EAE), an accepted animal model for multiple sclerosis. Both the proliferative functions (DNA synthesis and cell blastogenesis) and the EAE transfer activities of GPMBP-reactive lymphocytes were examined following GPMBP-induced activation in the presence of agents shown to block the outwardly rectifying K⁺ current in these cells. At concentrations which completely inhibited DNA synthesis, as measured by [³H]thymidine incorporation, and cell blastogenesis, tetraethylammonium (TEA), 4-aminopyridine (4-AP) and methoxyverapamil (D600) completely blocked the subsequent adoptive transfer of EAE into naive syngeneic Lewis rats. The concentrations at which these blockers produced a 50% reduction in DNA synthesis were estimated to be 16, 1.6 and 32 µM for TEA, 4-AP and D-600, respectively, which were roughly equivalent to the EC₅₀ to block the K⁺ current. Apamine, a potent Ca²⁺-activated K⁺ channel blocker, at a concentration several orders of magnitude higher than is necessary to block Ca²⁺-activated K⁺ channels, reduced the maximal K⁺ conductance in GPMBP-reactive T cell K⁺ channels by about 20%, but did not alter either [³H]thymidine incorporation or the adoptive transfer of EAE. These results indicate that delayed rectifier K⁺ channel blockers may prevent the activation of GPMBP-reactive T cells, thus prohibiting encephalitogenic effector cell functions.     

Role of the 30S ribosomal subunit, initiation factors, and specific ion concentration in barotolerant protein synthesis in Pseudomonas bathycetes.

Washed (1 M NH4Cl) ribosomes from Pseudomonas bathycetes, Pseudomonas fluorescens, and Escherichia coli were tested for their ability to synthesize protein or polypeptide at high pressure when used as such, when recombined with homologous initiation factors, and when recombined with heterologous initiation factors. The responses of natural messenger ribonucleic acid (MS-2)-directed systems to pressure were independent of the source of initiation factors and paralleled those of the washed ribosomes in polyuridylate-directed systems. In all cases, the responses to pressure were parallel to those obtained when unwashed ribosomes were utilized; therefore, we concluded that the initiation factors were interchangeable among these organisms, and that these factors did not play a critical role in determining the pressure responses of the protein-synthesizing systems. P. bathycetes ribosomal subunits were isolated under a variety of ionic conditions. These were tested for their ability to synthesize protein and polyphenylalanine at a variety of pressures when used in reconstituted P. bathycetes homologous systems and in hybrid systems with ribosomal subunits from E. coli and P. fluorescens. O. bathycetes 30S subunits, isolated in a buffer solution containing 0 mM NaCl and O mM KC] were functional at any pressure; those isolated in the presence of 150 mM NaCl and 0 mM KCl were functional at 1 atmosphere but barosensitive, and those isolated in the presence of O mM NaCl and 150 mM KCl retained the ion-mediated barotolerance characteristic of crude P. bathycetes ribosome preparations. The 50S subunit remained functional regardless of the method of isolation, and it had no effect on pressure sensitivity.     

Identification and quantification of proteins differentially secreted by a pair of normal and malignant breast‐cancer cell lines

Secreted proteins play a pivotal role in cellular functions. To better understand malignant behavior, we adapted stable isotopic labeling with amino acids in cell culture technology to identify and quantify proteins differentially released into the extracellular media by a pair of normal and malignant breast‐cancer cell lines. Approximately 380 non‐redundant proteins were quantified in serum‐free media. Of the assigned proteins, 62% are classified secreted in protein databases and an additional 25% are designated secreted in the literature. A number of growth factors were found differentially regulated. Tumor necrosis factor, pigment epithelial‐differentiating factor and stem‐cell growth factor precursor showed decreased expression in breast‐cancer cell line, whereas Inhibin beta and macrophage migration inhibitory factor show increased expression. Interestingly, protease inhibitors, including plasma protease (C1) inhibitor, PZP precursor, and SerpinE2 were significantly down‐regulated in cancer cell line as were angiostatic factors from extracellular matrix (ECM) such as endorepillin. Further, the C‐terminal fragment of type XVIII collagen, endostatin, a potent angiostatic factor, was down‐regulated as well whereas extracellular collagens and osteoblast‐specific factor 2 (OSF‐2), were up‐regulated. Differential expression and secretion of SerpinE2 and OSF‐2 were confirmed using Western blotting. These results corroborate models of invasive tumors sustained by elaborate coordination of stromal cells via chemokines and growth factors, while protease inhibitors remodel the ECM to stimulate angiogenesis.     

Reversal of motility loss in bongo antelope (Tragelaphus eurycerus isaaci) spermatozoa contaminated with hyposmotic urine during electroejaculation

Semen collected by a combination of ampullary (rectal) massage and electroejaculation of a bongo bull was incidentally contaminated with urine (1:3.7). At 1.5h post-collection, progressive motility was 0% but some spermatozoa had intermittently twitching tails. Subsequent dilution with media and processing improved the progressive motility (up to 50%) and intact membranes (up to 71%) of spermatozoa. After thawing, the respective values were 35 and 70%. The osmolarity and pH of the contaminated supernatant was 151 mOsm and 7.45, respectively. Initial progressive motility in a non-contaminated portion of semen collected during the same procedure was 80%, and, after thawing, 60 and 90%, of the spermatozoa showed progressive motility and intact membranes, respectively. In conclusion, urine-contaminated bongo spermatozoa can regain progressive motility after dilution with isosmotic solutions and survive cryopreservation.     

Cherty carbonate facies of the Montoya Group, southern New Mexico and western Texas and its regional correlatives: a record of Late Ordovician paleoceanography on southern Laurentia

The Upper Ordovician Montoya Group in southern New Mexico and westernmost Texas records predominantly subtidal deposition on a gently dipping carbonate ramp that was subsequently nearly entirely dolomitized. The medial unit of the Montoya Group, the Aleman Formation is unique because it contains abundant chert (10–70% by volume). The chert occurs as: (1) thin continuous beds of sponge spicules within mudstone or calcisiltite; (2) discontinuous, lenses or nodules within skeletal wackestones and packstones; or (3) as a replacement of skeletal grains and burrows. Coeval skeletal grainstones and muddy peritidal facies contain little chert. Phosphate (up to 5 wt.%) occurs within the underlying Upham Formation and the Aleman Formation as replacement of fossils and peloids. The abundance of chert and phosphate in these subtidal facies indicates they formed within a region of strong upwelling. Regional correlation with Upper Ordovician cherty units along the periphery of southern Laurentia and other low latitude continents suggests that upwelling was widespread and long-lived during the Late Ordovician. The upwelling is interpreted to record vigorous oceanic circulation produced by the onset of glaciation on Gondwana during this period. Late Ordovician relative sea-level curves around the periphery of Laurentia indicate correlative third-order (1–3 my duration) fluctuations that may provide a means for high-resolution global correlations. However, the mechanism(s) that produced these long-term fluctuations are unclear.     

Isolation by distance and gene flow in the Eurasian badger (Meles meles) at both a local and broad scale

Eurasian badgers, Meles meles, have been shown to possess limited genetic population structure within Europe; however, field studies have detected high levels of philopatry, which are expected to increase population structure. Population structure will be a consequence of both contemporary dispersal and historical processes, each of which is expected to be evident at a different scale. Therefore, to gain a greater understanding of gene flow in the badger, we examined microsatellite diversity both among and within badger populations, focusing on populations from the British Isles and western Europe. We found that while populations differed in their allelic diversity, the British Isles displayed a similar degree of diversity to the rest of western Europe. The lower genetic diversity occurring in Ireland, Norway and Scotland was more likely to have resulted from founder effects rather than contemporary population density. While there was significant population structure (F ST = 0.19), divergence among populations was generally well explained by geographic distance (P < 0.0001) across the entire range studied of more than 3000 km. Transient effects from the Pleistocene appear to have been replaced by a strong pattern of genetic isolation by distance across western Europe, suggestive of colonization from a single refugium. Analysis of individuals within British populations through Mantel tests and spatial autocorrelation demonstrated that there was significant local population structure across 3-30 km, confirming that dispersal is indeed restricted. The isolation by distance observed among badger populations across western Europe is likely to be a consequence of this restricted local dispersal.     

Proteolysis of mitotic chromosomes induces gradual and anisotropic decondensation correlated with a reduction of elastic modulus and structural sensitivity to rarely cutting restriction enzymes

The effect of nonspecific proteolysis on the structure of single isolated mitotic newt chromosomes was studied using chromosome elastic response as an assay. Exposure to either trypsin or proteinase K gradually decondensed and softened chromosomes but without entirely eliminating their elastic response. Analysis of chromosome morphology revealed anisotropic decondensation upon digestion, with length increasing more than width. Prolonged protease treatment resulted only in further swelling of the chromosome without complete dissolution. Mild trypsinization induced sensitivity of chromosome elasticity to five- and six-base-specific restriction enzymes. These results, combined with previous studies of effects of nucleases on mitotic chromosome structure, indicate that mild proteolysis gradually reduces the density of chromatin-constraining elements in the mitotic chromosome, providing evidence consistent with an anisotropically folded "chromatin network" model of mitotic chromosome architecture.     

Production of polysaccharides by callus cultures of common duckweed

Callus lines of common duckweed produced acid arabinogalactan and pectin in an amount varying from 1 to 3% of dry weight. The arabinogalactan specimens from the cell lines studied displayed a similar monosaccharide composition. The duckweed callus lines whose arabinogalactans contained apiose residues (1-2%) were found. All pectin specimens had a similar qualitative monosaccharide composition but differed in the quantitative content of monosaccharide residues. The lines with high contents of galactose, arabinose, and apiose in pectin specimens were obtained. The total content of neutral monosaccharide residues in pectins varied from 26 to 50%.