Parental DNA strands segregate randomly during embryonic development of Caenorhabditis elegans

The fate of gamete DNA was followed in the next generation embryos of the nematode C. elegans. Either male worms or spermless hermaphrodites were grown on bromodeoxyuridine-containing E. coli in order to label germ-line DNA. Matings then produced embryos in which only the DNA strands provided by the gametes contained label. This original gamete DNA could be detected during embryonic development by using a fluorescently labeled monoclonal antibody specific to bromodeoxyuridine. Both the number and position of fluorescent spots in the embryo indicate that gamete DNA strands segregate randomly during development. Random segregation of parental DNA strands rules out models of development that invoke chromosome imprinting or immortal DNA strands.     

The major gut esterase locus in the nematode Caenorhabditis elegans

Summary Mutations in the major gut esterase of the nematode Caenorhabditis elegans have been induced by ethylmethane sulfonate and detected by isoelectric focusing. The gut esterase locus, denoted ges-1, maps less than 0.3 map units to the right of the unc-60 locus, at the left end of chromosome V.     

Recombinant murine IL-5 induces high rate IgA synthesis in cycling IgA-positive Peyer's patch B cells

Recent studies have shown that purified IL-5 from T cell lines and clones enhances IgA synthesis in LPS-triggered splenic B cell cultures, and that this effect is augmented by IL-4. In this study we have examined the ability of rIL-5 and rIL-4 to support spontaneous Ig synthesis in normal Peyer's patch (PP) B cell cultures. The rIL-4 supported proliferation of the HT-2 and in vivo adapted BCL-1 cell lines, increased Ia expression on normal spleen B cells, co-stimulated splenic B cell proliferation in the presence of anti-mu and enhanced IgG1 synthesis in LPS triggered splenic B cell cultures. The rIL-5 supported BCL-1 proliferation, co-stimulated splenic B cell proliferation in the presence of dextran sulfate, and increased IgA synthesis in LPS-stimulated splenic B cell cultures. Markedly enhanced IgA responses occurred in PP B cell, but not splenic B cell cultures supplemented with rIL-5 in the absence of an added B cell trigger. However, rIL-4 alone did not enhance IgA synthesis or increase the IgA synthesis of PP B cell cultures stimulated with rIL-5. The rIL-5 receptive PP B cells were present in the blast cell subpopulation, inasmuch as a low density fraction isolated on Percoll gradients accounted for the enhanced IgA synthesis. Further, cell cycle analysis of whole PP B cells using propidium iodide in conjunction with staining for surface B220, demonstrated that approximately 12 to 16% of the B cells were in the S and G2/M stages of cell cycle, the remainder being in Go + G1. The surface IgM+ B cells were predominantly in Go + G1, whereas the sIgA+ B cell subpopulation was enriched for cells in the S and G2/M compartments. The PP B cell subset responsible for enhanced IgA synthesis in the presence of rIL-5 was sIgA-positive because FACS-depletion of the sIgA+ B cells resulted in the total loss of rIL-5 enhanced IgA synthesis. Further, when PP B cells were enriched for sIgA+ B cells by cell sorting, these cells responded to rIL-5 with increased IgA synthesis in a dose-dependent manner. When the actual numbers of IgA secreting cells were assessed in PP B cell cultures with supplemental rIL-5, no significant increase in total IgA-producing cells was noted when compared with B cells cultured without rIL-5.(ABSTRACT TRUNCATED AT 400 WORDS)     

Modulation of IgM secretion and H chain mRNA expression in CH12.LX.C4.5F5 B cells by adrenocorticotropic hormone

The murine B cell line CH12.LX.C4.5F5 (CH12 (5F5) expresses adrenocorticotropin (ACTH) receptors, which can modulate IgM secretion by these cells. Interestingly, the response to ACTH was concentration dependent, inducing IgM secretion at subnanomolar amounts and suppressing secretion at micromolar amounts. With the use of an enzyme-linking immunospot assay it was possible to demonstrate that the ACTH-induced increase in IgM secretion by CH12 (5F5) cells was caused at least in part by an increase in the number of cells secreting IgM. CH12 (5F5) cells activated with suboptimal concentrations of LPS demonstrated a similar biphasic response. ACTH at concentrations of 10(-13) to 10(-9) M augmented IgM secretion in LPS-activated cells as much as sixfold, whereas 10(-6) M ACTH slightly decreased LPS-induced IgM secretion. At the mRNA level, subnanomolar concentrations of ACTH increased microH chain mRNA expression up to twofold in unstimulated or LPS-stimulated CH12 (5F5) cells. Taken together, these studies show that physiologically relevant concentrations of ACTH can interact directly with receptors on these B lymphocytes to enhance IgM secretion and microH chain mRNA expression. Although ACTH does increase intracellular cAMP levels in CH12 (5F5) B cells, it is unlikely that the induction of this second messenger pathway is by itself responsible for the ACTH induced B cell differentiation. The concentration of ACTH necessary to stimulate significant intracellular cAMP increases was 10- to 100-fold higher than that required to increase IgM secretion. Furthermore, CH12 (5F5) cells treated with varying concentrations of 8-bromo cAMP or cholera toxin were inhibited in their ability to secrete IgM. These results strongly suggest that the enhancing effects of ACTH on CH12 (5F5) IgM secretion are via mechanisms independent of those mediated by cAMP.     

Analysis of Acidic Metabolites of Biogenic Amines in Bovine Retina and Vitreous and Aqueous Humour by Gas Chromatography-Negative Ion Chemical Ionisation Mass Spectrometry

Acidic metabolites of a number of biogenic amines have been identified and quantified by reaction with either acetic or propionic anhydride in the aqueous phase followed by extraction into ethyl acetate, esterification of carboxyl groups with ditrifluoromethylbenzyl bromide (DTFMBzBr), and then conversion of the remaining free hydroxyl groups to acetates. Subsequent analysis of these derivatives revealed that most (greater than 60%) of the ion current was carried by the ion resulting from the loss of DTFMBz from the molecular ion. This made the method highly specific and practical--limits of detection were established at approximately 200 pg with a potential limit of detection below the picogram level. This method establishes unequivocally that the metabolites of tyramine, dopamine, and adrenaline/noradrenaline (4-hydroxyphenylacetic acid, 3,4-dihydroxyphenylacetic acid, and dihydroxymandelic acid, respectively) are present in bovine retina and in vitreous and aqueous humour. In addition, high concentrations of the dopamine metabolite homovanillic acid were found in retina and vitreous, but not in aqueous humour. p-Hydroxymandelic acid, the acidic metabolite of p-octopamine/p-synephrine, was identified in vitreous and in aqueous humour.     

LPS regulation of the immune response: separate mechanisms for murine B cell activation by lipid A (direct) and polysaccharide (macrophage-dependent) derived from Bacteroides LPS

Lipopolysaccharide (LPS) derived from Bacteroides fragilis has been reported to stimulate mitogenic responses in spleen cell cultures from the classical LPS-hyporesponsive C3H/HeJ mouse strain; however, we have shown that purified splenic B cells from C3H/HeJ mice are hyporesponsive to phenol-water extracted LPS from B. fragilis ATCC 25285 (B-LPS). In the present study, B-LPS and its purified lipid A and polysaccharide components were tested for their ability to induce mitogenic and polyclonal IgM synthesis in spleen cell and purified splenic B cell cultures from classical LPS-responsive and -hyporesponsive mice. Mitogenic responses to B-LPS and E. coli K235 LPS(Ph) of whole spleen cells (2 X 10(5) cells/culture) or purified B cells (5 X 10(5) cells/culture) from classical LPS-responsive mouse strains (C3H/HeN, BALB/c, C57BL/6J, C57BL/10Sn, and DBA/2), F1 mice (derived from crosses between LPS responsive and C3H/HeJ mice), and classical LPS-hyporesponsive mice (C3H/HeJ and C57BL/10ScN) were high, intermediate, and low, respectively. When a higher number of whole spleen cells (5 X 10(5) cells/well) were cultured, B-LPS induced high mitogenic responses in C3H/HeN, intermediate responses in F1, and lower but significant responses in C3H/HeJ cultures. Similar results were obtained when polyclonal IgM synthesis was assessed in cultures containing 1 X 10(6) cells/culture. In contrast, the purified lipid A component of B-LPS failed to induce mitogenic responses in either whole spleen or purified B cell cultures. The addition of purified splenic B cells from C3H/HeJ mice to C3H/HeN or C3H/HeJ splenic adherent cells resulted in mitogenic responses to B-LPS, implying that the hyporesponsiveness to B-LPS seen in whole spleen cell cultures from C3H/HeJ mice at the lower cell concentration was due to limiting numbers of M phi. When splenic B cells and M phi from either C3H/HeN or C3H/HeJ mice were incubated with the lipid A or the polysaccharide moiety of B-LPS, lipid A induced mitogenic responses only in C3H/HeN cultures, whereas the polysaccharide moiety induced similar responses in both C3H/HeN and C3H/HeJ cultures. These results suggest that Bacteroides lipid A does not stimulate B cells from the classical LPS-hyporesponsive C3H/HeJ mouse strain, whereas the polysaccharide moiety of B-LPS is biologically active and mediates B cell stimulation via M phi.     

CMCase production by Spicellum roseum in liquid and solid culture

Summary CMCase was produced by 7 strains of Spicellum roseum in both liquid and wheat bran solid substrate cultures. No growth occurred above 35°C. Maximum enzyme production occurred at 30°C, whereas best enzyme activity occurred at pH 5.0 and 50°C. In liquid cultures of S. roseum, NRRL strains 13103, 13104, and 13106 produced activities of ca. 1.1, 1.5, and 1.5 mg glucose per hr/ml culture supernate at 1 week and 2.9, 1.5, and 2.1, respectively at 3 weeks compared to Trichoderma reesei NRRL 11236 (MCG77), which produced activities of 2.8 and 1.3 at 1 and 3 weeks.The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned.     

Potential Roles of CCR5+ CCR6+ Dendritic Cells Induced by Nasal Ovalbumin plus Flt3 Ligand Expressing Adenovirus for Mucosal IgA Responses

We assessed the role of CCR5⁺/CCR6⁺/CD11b⁺/CD11c⁺ dendritic cells (DCs) for induction of ovalbumin (OVA)-specific antibody (Ab) responses following mucosal immunization. Mice given nasal OVA plus an adenovirus expressing Flt3 ligand (Ad-FL) showed early expansion of CCR5⁺/CCR6⁺/CD11b⁺/CD11c⁺ DCs in nasopharyngeal-associated lymphoid tissue (NALT) and cervical lymph nodes (CLNs). Subsequently, this DC subset became resident in submandibular glands (SMGs) and nasal passages (NPs) in response to high levels of CCR-ligands produced in these tissues. CD11b⁺/CD11c⁺ DCs were markedly decreased in both CCR5^−/− and CCR6^−/− mice. Chimera mice reconstituted with bone marrow cells from CD11c-diphtheria toxin receptor (CD11c-DTR) and CCR5^−/− or CD11c-DTR and CCR6^−/− mice given nasal OVA plus Ad-FL had elevated plasma IgG, but reduced IgA as well as low anti-OVA secretory IgA (SIgA )Ab responses in saliva and nasal washes. These results suggest that CCR5⁺CCR6⁺ DCs play an important role in the induction of Ag-specific SIgA Ab responses.     

ROS Production and NF-κB Activation Triggered by RAC1 Facilitate WNT-Driven Intestinal Stem Cell Proliferation and Colorectal Cancer Initiation

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