Costs and benefits for water strider (Aquarius paludum) females of carrying guarding, reproductive males

We describe the mating system of Aquarius paludum insularis based on field observations and test hypotheses about the effects of body size, hunger level and post-copulatory guarding on reproductive performance. The mating sequence of this species was typical for temperate water striders, except that most oviposition was carried out by tandem pairs, most of which were submerged. Mate guarding continued until the end of oviposition, lasting up to 18.2h, which was much longer than that recorded for other species of water striders. Pair partners changed after oviposition. Extended contact guarding reduced female mobility. In the case of females that carried long-winged males, there was a significant reduction in speed and stride between tandem as opposed to single females. However, when short-winged males were carried, there was not a significant difference. Short-term foraging efficiency did not differ significantly between tandem and single females, and thus did not reflect the difference in mobility. Hunger level did not significantly affect female mating receptivity. Although the number of harassment bouts by unpaired males did not differ between single and tandem females, single females suffered significantly more harassment. Females were able to lay fertilized eggs for about 15 days after a single copulation, but they accepted long guarding and multiple mating during this period as well. The cost of resisting male mating attempts appears to be greater than the cost of carrying males.     

A new general method for the biosynthesis of stable isotope-enriched peptides using a decahistidine-tagged ubiquitin fusion system: An application to the production of mastoparan-X uniformly enriched with 15N and 15N/13C

A new strategy is described for the production of peptides enriched with stable isotopes. Peptides of interest are expressed in Escherichia coli (E. coli) cells as recombinant fusion proteins with Saccharomyces cerevisiae ubiquitin. This method yields as much as 30–100 mg/l of isotope-enriched fusion proteins in minimal media. A decahistidine tag attached to the N-terminus of ubiquitin enables a one-step purification of the fusion protein via Ni²⁺-chelating affinity chromatography. The ubiquitin moiety is then easily and specifically cleaved off by a protease, yeast ubiquitin hydrolase. Since this enzyme is also expressed at a high level in E. coli cells and can be purified in one step, the presented strategy has an advantage in view of costs over others that use commercially available proteases. In addition, since ubiquitin fusion proteins easily refold, the fusion protein can be expressed either in a soluble form or as inclusion bodies. This flexibility enables us to prepare peptides that are unstable in a soluble state in E. coli cells. As an example, the expression and the uniform stable isotope enrichment with ¹⁵N and/or¹³ C are described for mastoparan-X, a tetradecapeptide known to activate GTP-binding regulatory proteins. An amide group at the C-terminus of this peptide can also be formed by our method. The presented system is considered powerful for the stable isotope enrichment of short peptides with proton resonances that are too severely overlapped to be analyzed solely by proton NMR.     

Transesterification between triolein and stearic acid catalyzed by lipase in CO2 at various pressures

Transesterification between triolein and stearic acid catalyzed by lipase in pressurized CO2 at 50 °C was classified into three regions according to the pressure. Below 5 MPa which was the non-solvent region, the reaction was limited in the liquid triolein phase and the reaction rate was very slow. In the near critical region, from 5 to 10 MPa, the reaction rate was maximal at 5.9 MPa because of the stabilization of the enzyme-substrate complex. In the supercritical region, above 10 MPa, the reaction rate increased with an increase in pressure reflecting the increase in solubility of substrate in supercritical CO2 © Rapid Science Ltd. 1998     

Stimulation of in vitro hematopoiesis by a murine fetal hepatocyte clone through cell—cell contact

We have previously shown that a fetal liver-derived epithelial cell clone, FHC-4D2, could support hematopoiesis in vitro through its colony-stimulating factor (CSF) activities in a short-term culture. In this study, since FHC-4D2 cells were found capable of maintaining hematopoietic progenitors in the coculture for a long time, we examined how FHC-4D2 could exert hematopoietic supporting activity in a long-term culture by coculturing adult bone marrow (BM) cells or fetal liver (FL) cells on a monolayer of FHC-4D2 cells. This clone could maintain the colony-forming unit of granulocytes and macrophages (CFU-GM) of BM for ≥ 12 weeks under the coculture condition, but the fibroblastic cell clone from the fetal liver, FHC-4A3, could not support the survival of CFU-GM, even for 1 week. In addition to BM CFU-GM, the FHC-4D2 clone also supported the survival of FL CFU-GM, burst-forming unit of erythroid cells (BFUe), and colony-forming unit of mixed progenitors (CFU-Mix) for longer than 4 weeks. When BM cells were separated by a membrane filter from the FHC-4D2 cells in the coculture, the comparable number of CFU-GM was maintained at day 3, but virtually no hematopoietic progenitors were detected at the end of the first week. CFU-GM were present in both nonadherent and adherent cells to the FHC-4D2 cells at day 3 of the coculture, but at day 7, the adherent population contained greater number of CFU-GM. CFU-GM derived from the adherent cells formed larger colonies and contained more bipotential CFU-GM than the nonadherent population. When BM cells from mice given 5-fluorouracil were cocultured with FHC-4D2 cells under the limiting dilution condition, interleukin-3 (IL-3)-responsive CFU-GM were induced from immature hematopoietic progenitor cells that were otherwise unresponsive to IL-3. From these data we conclude that the FHC-4D2 clone could generate and maintain IL-3-responsive hematopoietic progenitors via close contact and that, in the fetal liver, the contact between hepatocytes and hematopoietic cells may be critically important in inducing the differentiation of resting, IL-3-unresponsive immature hematopoietic cells into CFU-GM (progenitors responsive to IL-3) and in triggering the self-renewal of CFU-GM. © 1994 Wiley-Liss, Inc.     

Proliferation of hematopoietic cell lines induced by a soluble factor derived from human squamous cell carcinomas of the head and neck

The supernatant of a cell line of squamous cell carcinoma of the head and neck (SCCHN), PCI-50, was previously shown to induce activation, promote proliferation and increase antitumor cytotoxicity of freshly purified human natural killer (NK) cells and CD4⁺ T lymphocytes [Arch Otolaryngol Head Neck Surg (1994) in press]. This supernatant was found also to promote the growth of a variety of hematopoietic cell lines, including Jurkat, THP-1, K562, NK-92 or Epstein-Barr-virus-transformed B cell lines. The Jurkat cell line was selected as a reporter cell in an 18-h proliferation assay established to measure the growth-promoting activity of PCI-50 supernatant. The presence of soluble tumor-derived factors able to induce proliferation of Jurkat cells was demonstrated in the supernatant produced by several other SCCHN cell lines but not in that produced by a gastric cancer cell line (HR) or renal cell carcinoma line (5117G8). The growth-promoting PCI-50 supernatant was shown to contain 28±0.5 pg/ml interleukin-6, (IL-6) in vitro but was negative for interferon , IL-1, IL-2, IL-4, tumor necrosis factor , granulocyte/macrophage-colony-stimulating factor and IL-12. The addition of any of these recombinant cytokines to Jurkat cell cultures did not significantly promote growth, while PCI-50 supernatant was consistently growth-stimulatory. This supernatant neither enhanced intracellular Ca²⁺ concentration in Jurkat cells nor induced up-regulation of activation antigens on the cell surface, although it supported growth of Jurkat cells in the absence of IL-2. The growth-promoting activity in the PCI-50 supernatant was acid-labile at pH 2 for 4 h, heat-resistant at 96 °C for 1 h and sensitive to treatments with trypsin and pepsin. Preincubation of the PCI-50 producer cells with tunicamycin or cyclohexamide reduced the level of growth-promoting activity in the supernatant. A partial purification of this activity was achieved using Amicon filtration, chromatography on concanavalin-A-Sepharose and then a hydroxyapatite column and high-pressure liquid chromatography gel filtration. The partially purified glycoprotein had a molecular mass of 50–70 kDa, as detemined by gel filtration.This work was supported in part by the Pathology Education and Research Foundation and American Cancer Society grant IM-696 to TLW     

Protective Capacity of L-Form Salmonella typhimurium against Murine Typhoid in C3H/HeJ Mice

L forms of Salmonella typhimurium LT2 conferred strong protection to a lethal challenge with its parental bacterium on innately hypersusceptible C3H/HeJ mice, and its minimal protective dose was approximately 150 L-forming units. Although L-form S. typhimurium was avirulent for C3H/HeJ mice, it multiplied slowly in both the liver and spleen with the maximal growth 2–3 weeks after immunization and thereafter it persisted in the liver until 24 weeks. Protective immunity began to work between 4 and 6 weeks after immunization, and it remained active as long as the L forms colonized the liver (until 24 weeks after immunization). Vaccination with the L form induced a population of T cells responding to L-form whole-cell lysate (WCL), while delayed-type hypersensitivity (DTH) to the extract of S. typhimurium was induced after the establishment of solid immunity. Moreover, neither T-cell responses nor DTH to heat-killed S. typhimurium was generated. In addition, antibody responses were elicited to WCL but not to heat-killed S. typhimurium. These results indicate that protection conferred by the L forms is attributable to the persistent colonization of the L forms rather than the presence of DTH, and also that Salmonella cytoplasmic antigens are involved in induction of immunological responses by vaccination with the L forms.     

Cytoplasmic Membrane-Associated Protein (CAP) Isolated from Streptococcus pyogenes: As a New Bacterial Superantigen

A protein isolated from the cytoplasmic membranes of Streptococcus pyogenes (cytoplasmic membrane-associated protein, CAP) stimulated human T cells in vitro to induce their mitogenic response. This CAP-induced T cell proliferation required the presence of nylon-adherent accessory cells (AC) of either autologous or allogeneic origin in the reaction mixtures. In addition, the reaction was inhibited by monoclonal antibodies (mAbs) against major histocompatibility complex (MHC) class II molecules, HLA-DR and -DQ, but not -DP. Human lymphoid cell lines positive for HLA-DR but not those lacking it were also effective as AC for the reaction. A binding test using fluorescein-labeled protein revealed that CAP bound to the adherent monocytes and HLA-DR⁺ but not to -DR^– lymphoid cell lines. The proliferative response of T cells to CAP was, however, not inhibited by the addition of the lysosomotrophic agent NH₄Cl to the reaction mixtures. These results suggest that the presentation of CAP by AC to human T cells is mediated through binding of the protein to the MHC class II molecules but without being processed in the AC. The proliferative response of T cells was also found to be inhibited by addition of anti-CD2, -CD3 or -T cell receptor (TcR) mAbs. A major population responding to CAP was CD3⁺4⁺8^– T cells. CAP also appears to stimulate T cells bearing Vβ8 sequences much more selectively than T cells bearing other Vβs. These results indicate that this streptococcal membrane protein, CAP, may be a new protein belonging to a group of bacterial superantigens.     

Expression of antisense nodulin-35 RNA in Vigna aconitifolia transgenic root nodules retards peroxisome development and affects nitrogen availability to the plant

A nodulin-35 (N-35) cDNA encoding nodule-specific uricase (EC 1.7.3.3.) was isolated from a Vigna aconitifolia (mothbean) root nodule cDNA library. Sequence analysis of Vigna uricase (VN-35) cDNA revealed 90% homology to that of soybean. The VN-35 cDNA was inserted in the antisense orientation downstream of the CaMV—35S promoter, and transgenic hairy roots were formed on Vigna plants using Agrobacterium rhizogenes . Infection with Bradyrhizobium (cowpea) gave rise to root nodules on transgenic hairy roots supported by the wild-type shoot. Expression of antisense VN-35 RNA was detected in transgenic nodules on individual roots using polymerase chain reaction (PCR). The nodules expressing antisense VN-35 RNA were smaller in size and showed lower uricase activity than nodules formed on the hairy roots transformed with a binary vector containing β-glucuronidase (GUS) gene (used as control), and the plants exhibited nitrogen deficiency symptoms. Ultrastructural analysis and immunogold labeling with antibody against soybean N-35 revealed that the growth of peroxisomes was retarded in transgenic nodules expressing antisense VN-35 RNA. These data suggest that a reduction in ureide biosynthesis limits the availability of symbiotically reduced nitrogen to the plant. The nodules of tropical legumes appear to be specialized in nitrogen assimilation and are developmentally controlled to produce and transport ureides.