Is the peacock's train an honest signal of genetic quality at the major histocompatibility complex?

Peacocks are a classic example of sexual selection, where females preferentially mate with males who have longer, more elaborate trains. One of the central hypotheses of sexual selection theory is that large or elaborate male 'ornaments' may signal high genetic quality (good genes). Good genes are thought to be those associated with disease resistance and as diversity at the major histocompatibility complex (MHC) has been shown to equate to superior immune responses, we test whether the peacock's train reveals genetic diversity at the MHC. We demonstrate via a captive breeding experiment that train length of adult males reflects genetic diversity at the MHC while controlling for genome-wide diversity and that peahens lay more, and larger, eggs for males with a more diverse MHC, but not for males with longer trains. Our results suggest that females are assessing and responding to male quality in terms of MHC diversity, but this assessment does not appear to be via train length, despite the fact that train length reflects MHC diversity.

Correlations between heterozygosity and measures of genetic similarity: implications for understanding mate choice.

There is currently considerable interest in testing the effects of genetic compatibility and heterozygosity on animal mate preferences. Evidence for either effect is rapidly accumulating, although results are not always clear-cut. However, correlations between mating preferences and either genetic similarity or heterozygosity are usually tested independently, and the possibility that similarity and heterozygosity may be confounded has rarely been taken into account. Here we show that measures of genetic similarity (allele sharing, relatedness) may be correlated with heterozygosity, using data from 441 human individuals genotyped at major loci in the major histocompatibility complex, and 281 peafowl (Pavo cristatus) individuals genotyped at 13 microsatellite loci. We show that average levels of allele sharing and relatedness are each significantly associated with heterozygosity in both humans and peafowl, that these relationships are influenced by the level of polymorphism, and that these similarity measures may correlate with heterozygosity in qualitatively different ways. We discuss the implications of these inter-relationships for interpretation of mate choice studies. It has recently become apparent that mating preferences for 'good genes' and 'compatible genes' may introduce discordant choice amongst individuals, since the optimal mate for one trait may not be optimal for the other, and our results are consistent with this idea. The inter-relationship between these measures of genetic quality also carries implications for the way in which mate choice studies are designed and interpreted, and generates predictions that can be tested in future research.

High degree of conservation of nuclear microsatellite loci in the genus Clusia.

In plants, microsatellites and their flanking DNA are rarely conserved across a whole genus, let alone other genera in the same family. Therefore, the possibility of using microsatellite primers developed for a species across a large number of plant species in the same genus is often limited. Remarkably, dinucleotide nuclear microsatellites developed for Clusia minor and for Clusia nemorosa amplified homologous microsatellites in species across the whole genus Clusia. In this present study, we report on the DNA sequence variation across the genus of 3 microsatellite loci with varying levels of variation. Compared over the species, there was a correlation between the lengths of the microsatellite loci. Interrupts occurred multiple times and did not seem to lead to the death of the microsatellite. These highly conserved markers will be useful for studying the variable reproductive systems in the genus Clusia.

Development of a time-resolved immunofluorometric assay for quantitation of mucosal and systemic antibody responses.

We developed a solid phase immunoassay that measured mucosal and systemic antibody responses from mice inoculated with either a staphylococcal enterotoxin B vaccine (SEBv) or noninfectious virus-like particles (VLP) of lentiviral origin. The assay used time-resolved fluorescence (TRF) with affinity-purified goat anti-mouse IgA and IgG conjugated to samarium and europium chelates, respectively. By employing these fluorogenic conjugates with different spectral emissions, IgA and IgG specific for SEB or VLP were readily detected in serum and saliva from mice inoculated intranasally. The TRF assay detected antigen-specific IgA in saliva 10 min after the addition of enhancement solution, while a conventional alkaline phosphatase-based assay for salivary IgA required 18 h after substrate addition. The TRF assay also provided a significantly higher signal-to-noise ratio and exhibited greater sensitivity. TRF assays detected both IgA and IgG in the same well, thereby reducing sample and reagent requirements.

Impact of landscape management on the genetic structure of red squirrel populations.

Landscape management practices that alter the degree of habitat fragmentation can significantly affect the genetic structure of animal populations. British red squirrels use "stepping stone" patches of habitat to move considerable distances through a fragmented habitat. Over the past few decades, the planting of a large conifer forest has connected groups of forest fragments in the north of England with those in southern Scotland. This "defragmentation" of the landscape has resulted in substantial genetic mixing of Scottish and Cumbrian genes in squirrel populations up to 100 kilometers from the site of the new forest. These results have implications for the conservation management of animal and plant species in fragmented landscapes such as those found in Britain.

Microtiter-based assay for evaluating the biological activity of ribosome-inactivating proteins.

A microtiter assay was developed to quickly measure the biological activity of ricin or other ribosome-inactivating proteins. Nuclease-treated rabbit reticulocyte lysate containing luciferase mRNA was used to measure toxin activity via inhibition of protein synthesis. The relative biological activity was determined by comparing luminescence levels in treated samples versus those of untreated controls. The amount of luciferase translated, as measured by luminescence, was inversely proportional to the toxin concentration. Linear dose response curves were generated for both class I and class II toxins. When compared to normal serum controls, specific antibody against ricin effectively inhibited ricin activity. The assay is suitable for efficient in vitro screening of therapeutics, as well as for identifying samples containing ribosome-inactivating proteins.

Clostridium perfringens iota-toxin: mapping of receptor binding and Ia docking domains on Ib.

Clostridium perfringens iota-toxin is a binary toxin consisting of iota a (Ia), an ADP-ribosyltransferase that modifies actin, and iota b (Ib), which binds to a cell surface protein and translocates Ia into a target cell. Fusion proteins of recombinant Ib and truncated variants were tested for binding to Vero cells and docking with Ia via fluorescence-activated cytometry and cytotoxicity experiments. C-terminal residues (656 to 665) of Ib were critical for cell surface binding, and truncated Ib variants containing > or = 200 amino acids of the C terminus were effective Ib competitors and prevented iota cytotoxicity. The N-terminal domain (residues 1 to 106) of Ib was important for Ia docking, yet this region was not an effective competitor of iota cytotoxicity. Further studies showed that Ib lacking just the N-terminal 27 residues did not facilitate Ia entry into a target cell and subsequent cytotoxicity. Five monoclonal antibodies against Ib were also tested with each truncated Ib variant for epitope and structural mapping by surface plasmon resonance and an enzyme-linked immunosorbent assay. Each antibody bound to a linear epitope within the N terminus (residues 28 to 66) or the C terminus (residues 632 to 655). Antibodies that target the C terminus neutralized in vitro cytotoxicity and delayed the lethal effects of iota-toxin in mice.

Clostridium perfringens iota toxin: binding studies and characterization of cell surface receptor by fluorescence-activated cytometry.

The binding characteristics of iota toxin, a binary enterotoxin produced by Clostridium perfringens type E, were studied by fluorescence-activated cytometry. The proteolytically activated binding component of iota toxin, iota b (Ib), bound to various cell types when incubated at 4, 25, or 37 degrees C for 10 min. The binding of Ib was inhibited by antisera against C. perfringens type E or Clostridium spiroforme culture supernatants, but not C. perfringens types C or D. Pretreatment of Vero cells with glycosidases or lectins did not affect Ib interactions, while pronase effectively prevented Ib binding to the cell surface. The Ib protomer (Ibp) bound to the cell surface, but trypsinization of Ibp was necessary for docking of the ADP-ribosylating component, iota a (Ia). Ia attached to cell-bound Ib within 10 min at 37 degrees C, but surface levels of Ia decreased 90% after 30 min and were undetectable by 60 min. Detectable surface levels of Ib also diminished over time, and Western blot analysis suggested internalization or embedment of Ib into the membrane.

Detection of Clostridium perfringens alpha toxin using a capture antibody ELISA.

Clostridium perfringens phospholipase C (PLC), commonly known as alpha toxin, is the lethal, dermonecrotic toxin produced by all strains and is considered a major virulence factor in clostridial myonecrosis. We developed a capture antibody ELISA that accurately and specifically quantitates alpha toxin produced by C. perfringens. Another PLC, derived from Bacillus cereus, and culture filtrates from various bacterial species including Clostridium bifermentans and Clostridium novyi were not cross-reactive in this ELISA. Standard curves generated with homogenous C. perfringens alpha toxin revealed detection limits of 19 ng/ml. The ELISA was more sensitive in detecting alpha toxin than techniques such as PLC enzymatic activity and mouse lethality assays.

Tissue compatibility of methylmethacrylate in cranial prostheses: a preliminary investigation.

An in vivo study using 48 disease-free male Lewis rats was conducted to determine the histologic difference between an alloplastic cranial prosthesis made with a monomer directly from the manufacturer and a triple-distilled monomer. The histologic difference in the tissue reaction between a cranial prosthesis sterilized with ethylene oxide and one sterilized with cobalt-60 irradiation was also evaluated. Histologic tissue biopsies of the cranium and brain tissues were obtained at 1, 3, 6, and 12 weeks. Tissue biopsies after the third week showed minimal inflammation and the microscopic findings were consistent with the reparative stage of wound healing. The findings also suggest that distillation of the monomer in heat-polymerized methyl-methacrylate is unnecessary for cranial prostheses. Cobalt-60 irradiation was found to be an effective alternative method of sterilization for such prostheses.

Interaction of lymphocytes and high endothelial venules in irradiated lymph nodes.

After total-body exposure to various doses of ionizing radiation, the ability of lymphocytes to interact specifically with high endothelial venules of rat cervical and mesenteric lymph nodes was analyzed in frozen sections. Following a radiation dose of 1.5 Gy, high endothelial venules remained intact and the binding of unirradiated lymphocytes to the venules was enhanced relative to unirradiated controls. At radiation doses above 5.0 Gy, damage to high endothelial venules was observed histologically as well as assessed functionally. There was a significant decrease in specific lymphocyte-venule binding and a significant increase in nonspecific binding. These findings suggest that radiation-induced damage to high endothelial venules might play a role in radiation-induced immunosuppression by interfering with the normal passage of lymphocytes from the blood into lymph nodes via a specific interaction between lymphocytes and high endothelial venules.

Further enrichment and analysis of rat CFU-s.

Using the monoclonal antibody W3/13, which recognizes a determinant expressed on a sialoglycoprotein, rat marrow cells with the phenotype Thy-1 antigen upper 20% positive (Ox720) and high molecular weight leukocyte common antigen negative (Ox22-) were separated into W3/13 dim (W3/13d) and W3/13 bright (W3/13b) subpopulations by single-laser cell sorting. The spleen colony-forming unit (CFU-s) was found in the W3/13d fraction. A 468-fold enrichment of CFU-s was achieved. Only 20% of the Ox720, Ox22-, and W3/13d cells were in the S phase of the cell cycle as compared to 56% of Ox720, Ox22-, and W3/13b cells. Using Indo-1, it was not possible to demonstrate increases in cytosolic Ca++ levels within the enriched CFU-s population by colony-stimulating factors (CSFs) or interleukins 1, 2, and 3. However, challenge with the Ca++ ionophore, ionomycin, demonstrated apparent heterogeneity of intracellular Ca++ management within the enriched CFU-s population. The source of this heterogeneity is not known. Only a 12-day CFU-s was detected in the rat, and it was predominantly, but not exclusively, a Rhodamine 123 (Rh123) dull cell.

Flow cytometry techniques in radiation biology.

Hematopoietic stem cells (HSC) are present in the marrow at a concentration of approximately 2-3 HSC per 1000 nucleated marrow cells. In the past, only clonogenic assays requiring 8-13 days and ten irradiated recipient rodents were available for assaying HSC. Because of the importance of HSC in the postirradiation syndrome, we have developed a new rapid method based on flow cytometry not only to assay but also to purify and characterize HSC. This new method makes extensive use of monoclonal antibodies conjugated to fluorescent phycobiliproteins through the sulfhydryls of the hinge region of the IgG molecule. An optical bench arrangement with a dye laser and an argon laser was used for dual excitation of the phycobiliprotein-monoclonal antibody conjugates and various cellular and DNA probes. Using 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) exclusion to identify viable cells, it was possible to follow regeneration of postirradiated rat marrow HSC.

Characterization of rat prothymocyte with monoclonal antibodies recognizing rat lymphocyte membrane antigenic determinants.

Utilizing the technique of fluorescence-activated cell sorting and monoclonal antibodies directed at rat membrane antigens, various subpopulations of Lewis bone marrow cells were isolated and subsequently transfused into sublethally irradiated, histocompatible NBr recipient rats by either intravenous or intrathymic inoculation. Recipient rats were sacrificed and cell suspensions from thymus and other lymphoid tissue were examined for the presence of the RT7.1 marker on Lewis thymus-derived lymphocytes by fluorescence-activated cell analysis. From these studies, the population of Lewis bone marrow cells that could reconstitute T cells in the NBr rats was found to be Ox-22 negative, Ox-7 positive, W3/13 positive, and Ox-18 positive. Further analysis characterized the prothymocyte as being Ox-7 upper 20% positive and W3/13 weakly positive. In addition this marrow cell population was able to protect lethally irradiated Lewis rats (9.5 Gy) in 30-day survival tests. These studies have indicated that the prothymocyte either has been derived from the Ox-22 negative, Ox-7 upper 20% positive, and W3/13 positive marrow cells or, like the hematopoietic stem cell, this cell has also been characterized by this phenotype.

Purification and analysis of rat hematopoietic stem cells by flow cytometry.

The monoclonal antibody OX7 recognizes an epitope expressed on the Thy-1 glycoprotein, OX22 recognizes the high molecular weight form(s) on leukocyte common antigen, and W3/13 recognized determinants found on certain sialoglycoproteins. Recently, the rat colony-forming unit spleen (CFU-S) was characterized as being OX7 upper 20% positive (OX7u20%), OX22 negative (OX22-), and W3/13 weakly positive (W3/13+). In the present study these observations have been extended to include the hematopoietic stem cell (HSC). Rat marrow cells were incubated with allophycocyanine-OX7 Fab' (APC-OX7 Fab') and phycoerythrin B-OX22 Fab' (Phy B-OX22 Fab'). The cells were sorted with a FACS-II instrument by using a Krypton laser tuned to the 530 nm spectral line for phycobiliprotein excitation. It was found that marrow cells capable of protecting lethally irradiated Lewis rats (9.5 Gy total body radiation, 0.4 Gy/min Co60) had the phenotype OXu20%, OX22-. The percentage of cells in the marrow with this phenotype was found to be 0.34 +/- 0.01 (mean +/- S.E.). Three thousand of these cells were required to rescue 50% of lethally irradiated recipients (30-d survival), while the number of unsorted bone marrow cells required was 1.05 X 10(6). Thus, a 350-fold purification of the HSC was realized. Although CFU-S copurified with HSC, purification of only 105-fold was obtained. This might indicate that purified HSC have a reduced capacity to generate splenic hematopoietic colonies. The OX7u20%, OX22- -enriched HSC population could be further divided into W3/13 dim and W3/13+ subpopulations by three-parameter immunofluorescence analysis with the use of a new optical bench arrangement.

Rat colony-forming unit spleen is OX7 positive, W3/13 positive, OX1 positive, and OX22 negative.

Using the monoclonal antibodies OX7 HL, W3/13 HLK, OX1 HLK, OX22, and the technique of fluorescence activated cell sorting, it was possible to characterize the phenotype of the rat marrow CFU-S as OX7 upper 20% positive, W3/13 lower 50% positive, OX1 positive, and OX22 negative. OX7 recognizes an antigenic determinant expressed on the Thy1 glycoprotein, W3/13 recognizes a determinant expressed on some sialoglycoproteins, OX1 recognizes all four apparent molecular weight forms of leukocyte common antigen, while OX22 recognizes only the high molecular weight forms of leukocyte common antigen. It was determined that the concentration of OX7 upper 20% positive, W3/13 lower 50% positive, OX1 positive, and OX22 negative cells in the marrow was 3085 +/- 1446/10(6) cells. For comparison, the calculated concentration of marrow stem cells using a 2-h seeding efficiency was found to be 501 CFU-S/10(6) cells and, using a 24-h seeding efficiency, it was found to be 1415 CFU-S/10(6) cells. Although requiring further refinements, these results suggest that an assessment of CFU-S marrow concentration might be achieved using multiparameter flow cytometry. Also, a technique for the conjugation of the Fab' fragment of the monoclonal antibody OX7 to phycoerythrin is described.

Effect of sublethal ionizing radiation on rat Peyer's patch lymphocytes.

After sublethal doses of ionizing radiation, rat Peyer's patch lymphocytes regenerated significantly more slowly than lymphocytes from spleen, thymus, and peripheral lymph nodes. Long Evans rats were exposed to 150 rad (40 rad/min) of whole-body irradiation from a 60Co, gamma-emitting source. On Days 1-20 postirradiation, single cell suspensions of lymphocytes from thymus, spleen, peripheral lymph nodes, and Peyer's patches were stained with mouse monoclonal antibody reagents specific for rat lymphocyte subpopulations (Ia+ cells, non-helper T-cell subsets, and helper T-cell subsets). Cells were then counterstained with Texas Red-conjugated, goat anti-mouse IgG and, at the same time, were also stained with fluorescein diacetate to determine viable lymphocytes. The stained lymphocytes were analyzed using a dual-laser, fluorescent-activated cell sorter (Becton-Dickinson FACS-II) from which the percentage of each lymphocyte subpopulation was determined. From our studies, we found that all subpopulations of lymphocytes were affected similarly by irradiation. In addition, we observed that viable lymphocyte subpopulation in thymus, spleen, and peripheral lymph nodes from irradiated animals returned to normal (nonirradiated control animals) levels 5-12 days postirradiation, while viable lymphocyte subpopulations in Peyer's patches from irradiated animals remained suppressed up to 20 days postirradiation. These results suggest that either the lymphocytes or, more likely, the microenvironment of Peyer's patches is more greatly damaged by ionizing radiation than that observed in other lymphoid tissue.

Effect of mouse type I interferon on mouse bone marrow cells and peritoneal exudate cells cultured in vitro.

Type I mouse interferon significantly reduces macrophage colony formation from mouse bone marrow cells or mouse peritoneal exudate cells when cultured in vitro in the presence of colony-stimulating factors (CSF) derived from either pregnant mouse uterus (CSFPMUE or LCM (CSFLCM) (concanavalin-A purified preparation from endotoxin-shocked mouse long-conditioned medium). The effect of interferon on granulocyte (G-CFU) or granulocyte-macrophage-mixed (GM-CFU) colonies depended on the CSF used. G-CFU and GM-CFU were somewhat elevated in interferon-treated cells stimulated by CSFPMUE whereas G-CFU and GM-CFU were slightly inhibited in interferon-treated cells stimulated by CSFLCM. The most dramatic effect of interferon was observed in the macrophage population. We conclude that interferon affects differentiation preferentially at the macrophage progenitor cell level.