IL-1 as adjuvant. Role of T cells in the augmentation of specific antibody production by recombinant human IL-1 alpha

Human rIL-1 alpha significantly enhanced splenic plaque-forming cells (PFC) to SRBC in vitro and in vivo. A single i.p. injection was sufficient to produce a fivefold or greater increase in the generation of PFC in a primary response. IL-1 treatment resulted in an increased production of Ag-specific PFC, both in vitro and in vivo, in combination with suboptimal doses of Ag. When IL-1 was given with a primary dose of Ag in vivo, an enhanced IgG response occurred. IL-1 enhanced in vivo carrier priming for an anti-hapten PFC response, indicating increased Th activity. Furthermore, T cells from spleens of mice treated with IL-1 provided significantly more help in both carrier (SRBC)- and hapten (TNP)- specific PFC. The enhancement of PFC by IL-1 in vitro occurred even in the presence of an excess of neutralizing anti-IL-2 antibody. These results suggest that IL-1 may enhance T cell-dependent antibody production in part by increasing Th activity, and that the mechanism of IL-1 action in increasing antibody production involves pathways in addition to the induction of IL-2 secretion.     

A method for cultivating morphologically undifferentiated embryonic stem cells from porcine blastocysts

Variable conditions were tested to determine an in-vitro cultivation method for the formation of morphologically undifferentiated embryonic stem cells from the inner cell mass (ICM) derived outgrowth of porcine blastocysts. Although all 16 Day-9 embryos failed to form colonies, 14 such colonies were obtained from a total of 69 Day-10 embryos when they were co-cultivated with porcine uterine fibroblast (PUF) cells over a 6-day period. The best results were obtained in Dulbecco's modified Eagle medium (DMEM) with 10% fetal calf serum and 10% porcine serum supplemented with bovine insulin and beta-mercaptoethanol, in which six out of seven embryos formed adequate ICM-derived colonies. Since murine fibroblasts were not found to be suitable feeder cells in this procedure, an endocrine synergistic interaction, which promotes embryonic attachment and colony formation, between porcine blastocysts and PUF cells is hypothesized. Continued propagation of the ICM-derived cells was not dependent on these factors; a total of seven cell lines were obtained after three to five subsequent passages on murine feeder-layers that resembled morphologically undifferentiated embryonic cells.     

An organic radical in the lysine 2,3-aminomutase reaction.

Lysine 2,3-aminomutase from Clostridium SB4 has been studied by electron paramagnetic resonance (EPR) spectroscopy at 77 K. Although the reaction catalyzed by this enzyme is similar to rearrangements catalyzed by enzymes requiring adenosylcobalamin, lysine 2,3-aminomutase does not utilize this cofactor. The enzyme instead contains iron-sulfur clusters, cobalt, and pyridoxal phosphate and is activated by S-adenosylmethionine. Subsequent to a reductive incubation procedure that is required to activate the enzyme, EPR studies reveal the appearance of an organic radical signal (g = 2.001) upon addition of both L-lysine and S-adenosylmethionine. The radical signal is complex, having multiple hyperfine transitions. The total radical concentration is proportional to enzyme activity and decreases in parallel with the approach to chemical equilibrium between alpha-lysine and beta-lysine. The signal changes over the time course of the reaction in a way that suggests the presence of more than one radical species, with different relative proportions of species in the steady state and equilibrium state. Isotopic substitution experiments show that unpaired spin density resides on the molecular framework of lysine and that solvent-exchangeable protons do not participate in strong hyperfine coupling to the radical. The results indicate that lysine radicals participate in the rearrangement mechanism.     

Conformational properties of the beta(400-436) and beta(400-445) C-terminal peptides of porcine brain tubulin.

Two peptides from the C-terminal region of the major beta-tubulin isotype (400-436 and 400-445) that include the critical areas for interaction with MAP2 and tau were examined to determine their conformations in aqueous solution. Despite a high theoretical potential for alpha-helix formation, CD spectroscopy showed that these peptides consisted primarily of random coil with some reverse turn. This was unaffected by the presence of counterions to the negatively charged side chains (Ca2+, Mg2+), but did change when the side-chain charges were neutralized by lowering the pH; under these conditions, the alpha-helix content of the longer peptide rose to 25% and the C-terminal truncated peptide to 15%. The peptides also adopt alpha-helical structure in the presence of trifluoroethanol, the truncated peptide again attaining a lower maximum percentage. The beta(400-445) peptide was also studied by 1-D and 2-D NMR techniques. The results indicate that at pH 5.6 or 7 in an aqueous solution the peptide is extremely flexible and lacks regular secondary structure, consistent with the CD results. Both peptides inhibited microtubule-associated protein-stimulated tubulin assembly, with the longer peptide being about 4 times as inhibitory as the smaller peptide. Neither was inhibitory in the absence of microtubule-associated proteins, indicating that interaction with this species was necessary for inhibition. The greater activity of the longer peptide could be due to the extra negative charges in this peptide and/or the greater tendency of this peptide to form an alpha-helical structure under the appropriate conditions.     

Genetic analyses of Rhizobium meliloti exopolysaccharides

We have recently obtained strong genetic evidence that the acidic Calcofluor-binding exopolysaccharide (EPS I) of Rhizobium meliloti Rm1021 is required for nodule invasion and possibly for later events in nodule development. Thirteen loci on the second megaplasmid have been identified that are required for, or affect, the synthesis of EPS I. Mutations in certain of these loci completely abolish the production of EPS I and result in mutants that form empty Fix- nodules. exoH mutants fail to succinylate their EPS I and form empty Fix- nodules. We have identified two unlinked regulatory loci, exoR and exoS, whose products play negative roles in the regulation of expression of the exo genes. We have recently discovered that R. meliloti has a latent capacity to synthesize a second exopolysaccharide (EPS II) that can substitute for the role(s) of EPS I in nodulation of alfalfa but not of other hosts. Possible roles for Rhizobium exopolysaccharides in nodulation are discussed.     

Temporal changes in the level of infestation of Simulium ornatum Meigen (complex) (Simuliidae: Diptera) larvae by the endosymbiotic fungus Harpella melusinae Lichtwardt (Harpellales: Trichomycetes)

The fungus Harpella melusinae (Harpellales: Trichomycetes) is obligately associated with the midguts of larval Simuliidae (Diptera). The level of infestation of a population of Simulium ornatum by H. melusinae was monitored at a stream in Hampshire, England. Significant temporal changes in the level of infestation were recorded during monthly and weekly collections; a twenty-fold increase being recorded over a nine-day period. Possible mechanisms by which these changes occur are discussed.     

Behavioral and hormonal aspects of reproduction in captive goeldi’s monkeys (Callimico goeldii) in a comparative and evolutionary context

The fate of most nonhuman primate species is intimately related to man. The increasing encroachment on the natural habitat has resulted in the decimation and even near extinction of some species. Along with this development, the basic concept in many modern zoos has changed from one of merely display to self-perpetuating units. Primate research facilities are orienting their research programs towards reproductive physiology and behavior in an effort to provide basic knowledge of reproduction in these species. This increased emphasis in the area of reproductive biology and the various efforts to improve breeding of these mostly endangered primates in captivity has stimulated the author to write this review. It represents an attempt to provide the reader with basic background information relating to the endocrinology and behavior of reproduction in the clawed New World monkeys as it exists at the time of publication. The intermediate evolutionary position ofCallimico goeldii between the clawed New World monkeys and the ‘true New World monkeys’ and our relatively poor knowledge about reproduction and behavior in this particular species fully justifies the focus on Goeldi’s monkey in this essay. This review is an attempt to provide a brief history of previous studies but also the basis for research in the future. The current status of knowledge of the small-bodied clawed monkeys is also discussed in an evolutionary context, with an emphasis on the different reproductive strategies in this dynamic group of primates. The outcome, not surprisingly, confirms the unique position ofCallimico goeldii in its social, ecological, and evolutionary environment.     

Properties of ganglioside GM1 in phosphatidylcholine bilayer membranes.

Gangliosides have been shown to function as cell surface receptors, as well as participating in cell growth, differentiation, and transformation. In spite of their multiple biological functions, relatively little is known about their structure and physical properties in membrane systems. The thermotropic and structural properties of ganglioside GM1 alone and in a binary system with 1,2-dipalmitoyl phosphatidylcholine (DPPC) have been investigated by differential scanning calorimetry (DSC) and x-ray diffraction. By DSC hydrated GM1 undergoes a broad endothermic transition TM = 26 degrees C (delta H = 1.7 kcal/mol GM1). X-ray diffraction below (-2 degrees C) and above (51 degrees C) this transition indicates a micellar structure with changes occurring only in the wide angle region of the diffraction pattern (relatively sharp reflection at 1/4.12 A-1 at -2 degrees C; more diffuse reflection at 1/4.41 A-1 at 51 degrees C). In hydrated binary mixtures with DPPC, incorporation of GM1 (0-30 mol%; zone 1) decreases the enthalpy of the DPPC pretransition at low molar compositions while increasing the TM of both the pre- and main transitions (limiting values, 39 and 44 degrees C, respectively). X-ray diffraction studies indicate the presence of a single bilayer gel phase in zone 1 that can undergo chain melting to an L alpha bilayer phase. A detailed hydration study of GM1 (5.7 mol %)/DPPC indicated a conversion of the DPPC bilayer gel phase to an infinite swelling system in zone 1 due to the presence of the negatively charged sialic acid moiety of GM1. At 30-61 mol % GM1 (zone 2), two calorimetric transitions are observed at 44 and 47 degrees C, suggesting the presence of two phases. The lower transition reflects the bilayer gel --> L alpha transition (zone 1), whereas the upper transition appears to be a consequence of the formation of a nonbilayer, micellar or hexagonal phase, although the structure of this phase has not been defined by x-ray diffraction. At > 61 mol % GM1 (zone 3) the calorimetric and phase behavior is dominated by the micelle-forming properties of GM1; the presence of mixed GM1/DPPC micellar phases is predicted.     

Induction of paternal genome loss by the paternal-sex-ratio chromosome and cytoplasmic incompatibility bacteria (Wolbachia): A comparative study of early embryonic events

Paternal genome loss (PGL) during early embryogenesis is caused by two different genetic elements in the parasitoid wasp, Nasonia vitripennis. Paternal sex ratio (PSR) is a paternally inherited supernumerary chromosome that disrupts condensation of the paternal chromosomes by the first mitotic division of fertilized eggs. Bacteria belonging to the genus Wolbachia are present in Nasonia eggs and also disrupt paternal chromosome condensation in crosses between cytoplasmically incompatible strains. Cytoplasmic incompatibility Wolbachia are widespread in insects, whereas PSR is specific to this wasp. PGL results in production of male progeny in Nasonia due to haplodiploid sex determination. The cytological events associated with PGL induced by the PSR chromosome and by Wolbachia were compared by fluorescent light microscopy using the fluorochrome Hoescht 33258. Cytological examination of eggs fertilized with PSR-bearing sperm revealed that a dense paternal chromatin mass forms prior to the first metaphase. Quantification of chromatin by epifluorescence indicates that this mass does undergo replication along with the maternal chromatin prior to the first mitotic division but does not replicate during later mitotic cycles. Contrary to previous reports using other staining methods, the paternal chromatin mass remains condensed during interphase and persists over subsequent mitotic cycles, at least until formation of the syncytial blastoderm and cellularization, at which time it remains near the center of the egg with the yolk nuclei. Wolbachia-induced PGL shows several marked differences. Most notable is that the paternal chromatin mass is more diffuse and tends to be fragmented during the first mitotic division, with portions becoming associated with the daughter nuclei. Nuclei containing portions of the paternal chromatin mass appear to be delayed in subsequent mitotic divisions relative to nuclei free of paternal chromatin. Crosses combining incompatibility with PSR were cytologically similar to Wolbachia-induced PGL, although shearing of the paternal chromatin mass was reduced. Wolbachia may, therefore, block an earlier stage of paternal chromatin processing in the fertilized eggs than does PSR. © 1995 Wiley-Liss, Inc.