Inhibition of L- and P-selectin by a rationally synthesized novel core 2-like branched structure containing GalNAc-Lewisx and Neu5Acalpha2- 3Galbeta1-3GalNAc sequences

The selectins interact in important normal and pathological situations with certain sialylated, fucosylated glycoconjugate ligands containing sialyl Lewisx(Neu5Acalpha2-3Galbeta1-4(Fucalpha1-3)GlcN Ac). Much effort has gone into the synthesis of sialylated and sulfated Lewisxanalogs as competitive ligands for the selectins. Since the natural selectin ligands GlyCAM-1 and PSGL-1 carry sialyl Lewisxas part of a branched Core 2 O-linked structure, we recently synthesized Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-6(SE-3Galbeta1++ +-3)GalNAc1alphaOMe and found it to be a moderately superior ligand for L and P-selectin (Koenig et al. , Glycobiology 7, 79-93, 1997). Other studies have shown that sulfate esters can replace sialic acid in some selectin ligands (Yeun et al. , Biochemistry, 31, 9126-9131, 1992; Imai et al. , Nature, 361, 555, 1993). Based upon these observations, we hypothesized that Neu5Acalpha2-3Galbeta1-3GalNAc might have the capability of interacting with L- and P-selectin. To examine this hypothesis, we synthesized Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-6(Neu5Acalpha2++ +-3Galbeta1-3)- GalNAc alpha1-OB, which was found to be 2- to 3-fold better than sialyl Lexfor P and L selectin, respectively. We also report the synthesis of an unusual structure GalNAcbeta1-4(Fucalpha1- 3)GlcNAcbeta1-OMe (GalNAc- Lewisx-O-methyl glycoside), which also proved to be a better inhibitor of L- and P-selectin than sialyl Lewisx-OMe. Combining this with our knowledge of Core 2 branched structures, we have synthesized a molecule that is 5- to 6-fold better at inhibiting L- and P-selectin than sialyl Lewisx-OMe, By contrast to unbranched structures, substitution of a sulfate ester group for a sialic acid residue in such a molecule resulted in a considerable loss of inhibition ability. Thus, the combination of a sialic acid residue on the primary (beta1-3) arm, and a modified Lexunit on the branched (beta1-6) arm on an O-linked Core 2 structure generated a monovalent synthetic oliogosaccharide inhibitor superior to SLexfor both L- and P-selectin.      

Beneficial effect of fluorocarbon emulsion media on the function of neuromuscular preparations in vitro

The effects of liquid fluorocarbons as bathing media were determined by use of in vitro neuromuscular preparations. Rat hemidiaphragms were bathed in either oxygenated fluorocarbon (FC) emulsion or standard oxygenated Krebs solution. Contractile force in response to simple supramaximal nerve stimuli as well as to high frequency stimulation was greater, while twitch:tetanus ratio was smaller in FC emulsion. With such medium, post-tetanic potentiation of contraction was also more consistently observed. Indirectly stimulated diaphragms survived longer in FC emulsion. After cessation of oxygenation, oxygen tension (ρO(2)) of the medium declined more rapidly with Krebs than with FC emulsion; ρO(2) directly correlated with force of contraction. Similarly, in the chick biventer cervicis preparation, FC emulsion enhanced nerve-stimulated force of contraction; returning the preparation to standard Krebs solution reversed this phenomenon. Dose-resonse curves of muscle contraction in response to acetycholine and KCl administration were shifted upward during FC emulsion superfusion. Frequency of miniature endplate potentials was lower in FC emulsion than that observed in Krebs solution, measured from the same cell of the rat diaphragm. Resting membrane potentials were also greater in muscle cells sampled from FC emulsion-bathed preparations. These data suggest that FC emulsion is superior to standard Krebs solution as a bathing medium for in vitro neuromuscular preparations by virtue of the high solubility of oxygen in it.     

Cellular organization of Bacillus subtilis: sodium dodecyl sulfate-induced cell partitioning into zebra structures.

Cells of Bacillus subtilis heated in high concentrations of sodium dodecyl sulfate (5%) and then washed free of detergent with a hot salt solution (80 C) become structurally reorganized into regions of densely compacted cytoplasm (termed zebras) and regions of sparsely filled material (termed spaces). Size distribution studies of zebras indicate that division-suppressed mutants and wild-type cells both yield zebras of comparable length. Similarly the lengths of zebras found in populations emerging from spores are uniform in one-, two-, three-, and four-zebra-containing cells. In contrast, the length of spaces is slightly larger than that of zebras and is unusually large in two-zebra-containing cells. The locations of zebras and spaces along cell length have been studied in spore out-growth populations. A statistical procedure developed previously in genome location investigations was used to analyze the location of zebras along cell length. The data indicate that as cells elongate, new sites arise where the cell contents are strongly bound to the cell surface. Within filament populations produced by division-suppressed mutants there is a linear relationship of mean filament length and zebra number per filament. These data indicate that cytoplasm in filaments with no obvious structural compartmentalizations may be organized into units associated with particular regions of cell surface. The attachment of cell contents to the cell surface may involve deoxyribonucleic acid. Zebra-containing cells digested with proteolytic enzyme and ribonuclease are converted to cells that contain a crystalline-like granule fixed at the location of each zebra. Exposure to deoxyribonuclease mobilizes these granules within the cell wall.     

A general model of reaction kinetics in biological systems

Dynamic mathematical models in biotechnology require, besides the information about the stoichiometry of the biological reaction system, knowledge about the reaction kinetics. Modulation phenomena like limitation, inhibition and activation occur in different forms of competition with the key enzymes responsible for the respective metabolic reaction steps. The identification of a priori unknown reaction kinetics is often a critical task due to the non-linearity and (over-) parameterization of the model equations introduced to account for all the possible modulation phenomena. The contribution of this paper is to propose a general formulation of reaction kinetics, as an extension of the Michaelis-Menten kinetics, which allows limitation/activation and inhibition effects to be described with a reduced number of parameters. The versatility of the new model structure is demonstrated with application examples.     

Parasite-mediated selection in experimental metapopulations of Daphnia magna

In metapopulations, only a fraction of all local host populations may be infected with a given parasite species, and limited dispersal of parasites suggests that colonization of host populations by parasites may involve only a small number of parasite strains. Using hosts and parasites obtained from a natural metapopulation, we studied the evolutionary consequences of invasion by single strains of parasites in experimental populations of the cyclical parthenogen Daphnia magna. In two experiments, each spanning approximately one season, we monitored clone frequency changes in outdoor container populations consisting of 13 and 19 D. magna clones, respectively. The populations were either infected with single strains of the microsporidian parasites Octosporea bayeri or Ordospora colligata or left unparasitized. In both experiments, infection changed the representation of clones over time significantly, indicating parasite-mediated evolution in the experimental populations. Furthermore, the two parasite species changed clone frequencies differently, suggesting that the interaction between infection and competitive ability of the hosts was specific to the parasite species. Taken together, our results suggest that parasite strains that invade local host populations can lead to evolutionary changes in the genetic composition of the host population and that this change is parasite-species specific.     

Tumor necrosis factor alpha induction of NF-kappaB requires the novel coactivator SIMPL

A myriad of stimuli including proinflammatory cytokines, viruses, and chemical and mechanical insults activate a kinase complex composed of IkappaB kinase beta (IKK-beta), IKK-alpha, and IKK-gamma/N, leading to changes in NF-kappaB-dependent gene expression. However, it is not clear how the NF-kappaB response is tailored to specific cellular insults. Signaling molecule that interacts with mouse pelle-like kinase (SIMPL) is a signaling component required for tumor necrosis factor alpha (TNF-alpha)-dependent but not interleukin-1-dependent NF-kappaB activation. Herein we demonstrate that nuclear localization of SIMPL is required for type I TNF receptor-induced NF-kappaB activity. SIMPL interacts with nuclear p65 in a TNF-alpha-dependent manner to promote endogenous NF-kappaB-dependent gene expression. The interaction between SIMPL and p65 enhances p65 transactivation activity. These data support a model in which TNF-alpha activation of NF-kappaB dependent-gene expression requires nuclear relocalization of p65 as well as nuclear relocalization of SIMPL, generating a TNF-alpha-specific induction of gene expression.     

The localisation of the extraneuronal monoamine transporter (EMT) in rat brain

The extraneuronal monoamine transporter plays an important role in the inactivation of monoamine transmitters. A basal extraneuronal tissue expression of this transporter has been reported, but it is also expressed in CNS glia. As little is known about the expression pattern and the function of the extraneuronal monoamine transporter in the brain, we performed a detailed investigation. Firstly, a northern blot analysis of different rat organs revealed that the transporter is strongly expressed in placenta, lung and heart and less prominently in the whole brain, brain stem, intestine, testis, epididymis, stomach, kidney and skeletal muscle. It was not expressed in cerebellum, liver and embryo. Using an in situ hybridization to the rat brain, we detected a marked and highly confined expression of the extraneuronal monoamine transporter in the area postrema, but in no other brain areas. These findings were confirmed by polyclonal antibodies against rat extraneuronal monoamine transporter showing an intensive signal in the area postrema, although a few cells in the cerebellum and the brain stem also showed a signal. Additionally, a partly overlapping expression pattern of the monoamine oxidase-B was detected. Summarizing, we firstly describe a marked and highly confined expression of the extraneuronal monoamine transporter in the rat area postrema by in situ hybridisation which may play a role in physiological functions of this circumventricular organ such as emesis, food intake and the regulation of cardiovascular functions.     

Synthesis and characterization of lanthanide orthothiophosphates: LaPS4

The single crystal structure of LaPS₄, (1), is reported. The space group is tetragonal, I4(1)/acd. Unit cell dimensions are a = 10.9641(3) Å and c = 19.4828(9) Å. The far infrared absorption and Raman spectra (100-600 cm⁻¹) are consistent with the groups being in a distorted tetrahedral geometry. The room temperature emission spectrum of LaPS₄ doped with Er³⁺ is also presented. Emission peaks at 529, 534, 549, and 554 nm were observed when the sample was excited at 492 nm. The compound reported here is isomorphous and isostructural to several other lanthanide orthothiophosphates.     

A panel of monoclonal antibodies recognizing GPI-anchored ADP-ribosyltransferase ART4, the carrier of the Dombrock blood group antigens

ART4 (CD297) is a member of the family of toxin-related ADP-ribosyltransferases (ARTs) and is the carrier of the Dombrock blood group alloantigens (Do). Two mouse monoclonal antibodies (MIMA-52 and MIMA-53), and two rat monoclonal antibodies (N0NI-B4 and NONI-B63) were obtained following immunization of mice with human Do/ART4-transfected cells and of rats with human Do/ART4 cDNA, respectively. All four mAbs recognize Do/ART4-transfected Jurkat cells but not untransfected cells by FACS analysis. Staining of Do/ART4-transfected cells by these mAbs was reduced following treatment of cells with PI-PLC, confirming that Do/ART4 is anchored in the cell membrane by linkage to glycosylphosphatidylinositol as predicted from its amino acid sequence. The four mAbs did not react with Gy(a-) (Dombrock null) erythrocytes but agglutinated other red blood cells. By flow cytometric analysis, all mAbs reacted prominently with erythrocytes, and weakly with peripheral blood monocytes and splenic macrophages, but not with B-lymphocytes or T-lymphocytes. The mAbs reacted weakly also with human umbilical vein endothelial cells and the basophilic leukemia KU-812. Immunohistology revealed staining of epithelia and endothelia on sections of tonsils. In FACS analyses NONI-B4 competed with MIMA-52 for binding to Do/ART4-transfected cells and erythrocytes, whereas NONI-B63 competed with MIMA-53. Neither of the mAbs reacted with mouse ART4-transfected cells, but NONI-B63 and MIMA-53 did react with a mouse/human ART4 chimera, indicating that the epitope recognized by these mAbs lies in the C-terminal half of the protein.     

Regulatory elements required for development of caenorhabditis elegans hermaphrodites are conserved in the tra-2 homologue of C. remanei, a male/female sister species

The Caenorhabditis elegans hermaphrodite is essentially a female that produces sperm. In C. elegans, tra-2 promotes female fates and must be repressed to achieve hermaphrodite spermatogenesis. In an effort to learn how mating systems evolve, we have cloned tra-2 from C. remanei, the closest gonochoristic relative of C. elegans. We found its structure to be similar to that of Ce-tra-2 but its sequence to be divergent. RNA interference demonstrates that Cr-tra-2 promotes female fates. Two sites of tra-2 regulation are required for the onset of hermaphrodite spermatogenesis in C. elegans. One, the MX region of TRA-2, is as well conserved in C. remanei as it is in C. briggsae (another male/hermaphrodite species), suggesting that this control is not unique to hermaphrodites. Another, the DRE/TGE element of the tra-2 3' UTR, was not detected by sequence analysis. However, gel-shift assays demonstrate that a factor in C. remanei can bind specifically to the Cr-tra-2 3' UTR, suggesting that this translational control is also conserved. We propose that both controls are general and do not constitute a novel "switch" that enables sexual mosaicism in hermaphrodites. However, subtle quantitative or qualitative differences in their employment may underlie differences in mating system seen in Caenorhabditis.