Immunosuppressive macromolecules of endometrial and conceptus origins in livestock species.

Since the mid- to late-1970s, intrauterine immunosuppressive macromolecules recovered from endometrial and conceptus secretions have been reported for livestock species. Using primarily in vitro assays, in conjunction with a limited number of techniques conducted in vivo, these macromolecules were shown to suppress various T-cell responses. Some macromolecules were also shown to suppress cytolytic activities of non T-cells. It remains unknown as to whether these macromolecules actually afford protection to the conceptus by suppressing cell-mediated immune responses directed toward conceptus tissues. Endometrial effector cells in the ewe respond to antigenic stimulation and preattachment trophoblastic cells of pigs and sheep can be lysed by effector cells. Consequently, these observations suggest a need for immunosuppression, either locally at placentation sites or within the entire uterus. This review describes the intrauterine macromolecules that have been shown to suppress lymphocyte responses. Additional information, although limited at this time, refers to their origin and possible mechanisms of action. As more reagents become available to complete the identification of the intrauterine immune cells in livestock animals, experiments (e.g. antibody-mediated depletion of cells) can be conducted to determine the precise functions of all these cells. Knowing their functions will help delineate whether or not immunosuppressive macromolecules have a role in the regulation and maintenance of conceptus tissues during pregnancy.

Identification and chromosomal localization of the monkey retrotransposon in Musa sp.

Retroelements are ubiquitous features of eukaryotic genomes, often accounting for a substantial fraction of their total DNA content. One major group of retroelements, which includes the gypsy and copia-like elements, is distinguished by the presence of long terminal repeats (LTRs). We have identified and partially characterized a sequence from banana (Musa acuminata cv. Grand Nain) which shows significant homology to gypsy-like LTR retroelements from other species. The element, named monkey, shows a high degree of homology to the reverse transcriptase, RNase H and integrase genes of retroelements from plants, fungi and yeast. However, several stop codons are present in the major ORF of this element, suggesting that this copy of monkey, if functional, is non-autonomous. Southern analysis indicated that monkey is present in both the A and B genomes of Musa, and that it is found in 200-500 copies per haploid genome in cv. Grand Nain. Chromosomal localization by fluorescent in-situ hybridization indicates that copies of monkey are concentrated in the nucleolar organizer regions and colocalize with rRNA genes. Other copies of monkey appear to be dispersed throughout the genome.

A porcine intrauterine 4-mda component with transforming growth factor-beta activity suppresses natural killer cell responses.

A 4-MDa component, recovered from uterine luminal secretions of gilts on d 15 of pregnancy, was assessed for suppression of the lytic responses from natural killer (NK) and lymphokine-activated killer (LAK) effector cells. Each cell type originated from preparations of peripheral blood lymphocytes (PBL), and the LAK cells were generated from the incubation of PBL with interleukin-2. The PBL and LAK cells were cultured for 5 d with and without the 4-MDa component. Following culture, the cells were incubated (22 h) with NK-sensitive K-562 target cells at varying effector:target cell ratios (25:1 to 200:1). Lytic activity was assessed with the chromium-51 release assay. Additional experiments were conducted in order to determine whether suppressor activity of the 4-MDa component was time-dependent and associated with transforming growth factor-beta2 (TGF-beta2). For effector:target cell ratios combined, the 4-MDa component suppressed the lytic activity of PBL but failed to affect the LAK cells. Suppression of NK-mediated lysis occurred by d 3 of the 5-d culture period. In addition, suppressor activity of the 4-MDa component was reversed by a neutralization antibody to TGF-beta2. In conclusion, the 4-MDa component with TGF-beta2 activity suppressed the lytic responses of porcine NK cells.

Suppressor activity of bone marrow cells and localization of fluorescent-labeled bone marrow cells within ovine endometrial tissue.

Numbers of fluorescein isothiocyanate (FITC)-labeled bone marrow (BM) cells of donor lambs were quantified within endometrial cell suspensions following their administration to ovariectomized (OVX; control-and estradiol-17beta-treated) and intact (estrus, d-14 cyclic and pregnant) ewes. The numbers of fluorescent BM cells were greater (P < .05) for the estrous and d-14 cyclic ewes than for both groups of OVX ewes. Fractionation of the endometrial cells with Percoll revealed that the majority of fluorescent cells were low-density (1.002 to 1.056 g/mL) cells. In coculture experiments, low-density cells from lamb BM not only suppressed the incorporation of thymidine into phytohemagglutinin-treated peripheral blood lymphocytes, but the cells also released suppressor factor into the culture medium. Suppressor activity tended to be reversed (P < .1) by a pan-specific neutralization antibody to transforming growth factor-beta (TGF-beta); however, the activity was unaffected by a neutralization antibody to TGF-beta2. These findings suggest that ovine endometrial suppressor cells may represent a population of low-density BM-derived natural suppressor cells, and their trafficking and localization patterns may depend on an ovarian factor(s). Further, suppressor activity does not seem to be mediated by TGF-beta2.

High-density ovine endometrial cells exhibit natural killer activity during early pregnancy.

Natural killer (NK)-like activity was assessed for peripheral blood lymphocytes (PBL) and unfractionated and fractionated endometrial cells recovered from ewes during the estrus cycle (Days 12 to 14) and early pregnancy (Days 16 to 18). The PBL and endometrial cells (each designated as effector cells) were cocultured with chromium-51 (51Cr) labeled NK-sensitive K-562 target cells in effector:target cell ratios ranging from 25:1 to 200:1, respectively. Lytic activity (i.e., release of 51Cr into the medium) was assessed at 22 h of culture. A high-density (> or = 1.088 g/mL) population of endometrial cells from the pregnant ewes exhibited NK-like activity, whereas endometrial cells from the cyclic ewes failed to exhibit activity. Lytic activity of these cells was greater (P < 0.05) for pregnant than for cyclic ewes (12.0 and 2.1%, respectively) at the effector:target cell ratio of 100:1, respectively. For both groups of ewes, PBL exhibited NK-like activity. These data indicate that the ovine endometrium contains NK-like cells with lytic activity between Days 16 and 18 of pregnancy.

Promoters derived from banana bunchy top virus DNA-1 to -5 direct vascular-associated expression in transgenic banana (Musa spp.).

The intergenic regions of banana bunchy top virus (BBTV) DNA-1 to -5 were fused to the green fluorescent protein (GFP) and uidA reporter genes and assessed for promoter activity in transgenic banana (Musa spp. cv. Bluggoe). Promoter activity associated with the BBTV-derived promoters was transgene dependent with greatest activity observed using the GFP reporter. The BBTV promoters (BT1 to BT5) directed expression primarily in vascular-associated cells, although levels of activity varied between individual promoters. Promoters BT4 and BT5 directed the highest levels of GFP expression, while activity from BT1, BT2 and BT3 promoters was considerably weaker. Intron-mediated enhancement, using the maize polyubiquitin 1 (ubi1) intron, generated a significant increase in GUS expression directed by the BBTV promoters in transgenic plants.

A tool for functional plant genomics: chimeric RNA/DNA oligonucleotides cause in vivo gene-specific mutations.

Self-complementary chimeric oligonucleotides (COs) composed of DNA and modified RNA residues were evaluated as a means to (i) create stable, site-specific base substitutions in a nuclear gene and (ii) introduce a frameshift in a nuclear transgene in plant cells. To demonstrate the creation of allele-specific mutations in a member of a gene family, COs were designed to target the codon for Pro-196 of SuRA, a tobacco acetolactate synthase (ALS) gene. An amino acid substitution at Pro-196 of ALS confers a herbicide-resistance phenotype that can be used as a selectable marker in plant cells. COs were designed to contain a 25-nt homology domain comprised of a five-deoxyribonucleotide region (harboring a single base mismatch to the native ALS sequence) flanked by regions each composed of 10 ribonucleotides. After recovery of herbicide-resistant tobacco cells on selective medium, DNA sequence analyses identified base conversions in the ALS gene at the codon for Pro-196. To demonstrate a site-specific insertion of a single base into a targeted gene, COs were used to restore expression of an inactive green fluorescent protein transgene that had been designed to contain a single base deletion. Recovery of fluorescent cells confirmed the deletion correction. Our results demonstrate the application of a technology to modify individual genetic loci by catalyzing either a base substitution or a base addition to specific nuclear genes; this approach should have great utility in the area of plant functional genomics.

Banana bunchy top virus DNA-2 to 6 are monocistronic.

Banana bunchy top virus (BBTV) DNA-3 to 6 have each previously been shown to contain one large open reading frame in the virion sense, whereas no large ORF had been identified in BBTV DNA-2. RNAs transcribed from the BBTV genome were mapped using northern hybridisation and 3' RACE. One mRNA was transcribed from each of BBTV DNA-2 to 6 and four of these mRNAs mapped to the ORFs previously identified in BBTV DNA-3 to 6. The mRNA of BBTV DNA-2 was transcribed from a virion sense ORF probably using a TATA box sequence different to that in BBTV DNA-1, and DNA-3 to 6. This ORF encoded a 10 kDa protein of unknown function. The 3' untranslated region of the five mRNAs varied from 25 nucleotides (BBTV DNA-6) to 167 nucleotides (BBTV DNA-4) and each contained putative polyadenylation signals with associated GT rich sequence together with a possible termination signal (C/T/A)TGTAA conserved in all five mRNAs.

Evaluation of natural Psoroptes ovis (Acarina: Psoroptidae) soluble proteins as candidate vaccine immunogens.

In this study potential vaccine candidate immunogens were identified and evaluated in a vaccine challenge trial. Calves vaccinated with a partially purified fraction of Psoroptes ovis-soluble proteins had 8 of 14 calves free of palpable lesions 8 wk after a challenge infestation. A self-grooming behavioral response elicited by a pruritic immediate-type allergic reaction was believed to be an effector in protecting the vaccinated calves from a clinical P. ovis infestation.

Promoter activity associated with the intergenic regions of banana bunchy top virus DNA-1 to -6 in transgenic tobacco and banana cells.

Promoter regions associated with each of the six ssDNA components of banana bunchy top virus (BBTV) have been characterized. DNA segments incorporating the intergenic regions of BBTV DNA-1 to -6 were isolated and fused to the uidA (beta-glucuronidase) reporter gene to assess promoter activity. In tobacco cell suspensions, the BBTV DNA-2 and -6 promoters generated levels of GUS expression 2-fold greater and similar to the 800 bp CaMV 35S promoter, respectively. Deletion analysis of the BBTV DNA-6 promoter suggested all the necessary promoter elements required for strong expression were located within 239 nucleotides upstream of the translational start codon. In transgenic tobacco plants, the BBTV-derived promoters generally provided a weak, tissue-specific GUS expression pattern restricted to phloem-associated cells. However, in callus derived from tobacco leaf tissue, GUS expression directed by the BBTV DNA-6 promoter was strong and, in some lines, comparable to the CaMV 35S promoter. Detectable promoter activity associated with the BBTV promoters in banana embryogenic cells was only observed using a sensitive green fluorescent protein (GFP) reporter. Promoters derived from BBTV DNA-4 and -5 generated the highest levels of transient activity, which were greater than that of the maize ubi-1 promoter. In transgenic banana plants, the activity of the BBTV DNA-6 promoter was restricted to the phloem of leaves and roots, stomata and root meristems.

Two mRNAs are transcribed from banana bunchy top virus DNA-1.

We have mapped the mRNA transcripts of banana bunchy top virus (BBTV) DNA-1. Northern hybridization and 3' RACE analysis identified two poly-adenylated RNAs associated with BBTV DNA-1. Previously, one major ORF in the virion sense of DNA-1 had been identified, which encoded a putative replication protein (Rep). An mRNA was identified in BBTV infected bananas that was clearly transcribed from this Rep ORF. Further, a second transcript was identified which mapped to an ORF completely within the Rep ORF. This encoded a putative 5 kDa protein of unknown function. Both these transcripts were also identified in a tobacco plant that had been transformed with Agrobacterium tumefaciens harbouring a binary construct containing the Rep ORF from BBTV DNA-1. This Rep ORF was inserted 3' of a cauliflower mosaic virus 35S promoter and 5' of a vegetable storage protein terminator. The transcripts mapped from these tobacco plants were identical at the 3' end to the transcripts from BBTV infected banana plants. The site of polyadenylation for the Rep ORF was at base 963 immediately 3' of the translational stop codon confirming that the polyadenylation signals for this transcript were all within the ORF. However, the internal ORF had a large untranslated region of 272 bases with its site of polyadenylation at nucleotide 803 and a polyadenylation signal 3' of the translational stop codon. A possible upstream termination signal (A/TTGTAA) was identified and was conserved within BBTV DNA-1 sequences from different international isolates.

Immunocytochemical localization of testis ecdysiotropin in the pupa of the gypsy moth, Lymantria dispar (L.) (Lepidoptera: Lymantriidae).

Antiserum against testis ecdysiotropin isolated from the gypsy moth, Lymantria dispar, reacted with neurons in the protocerebrum, optic and antennal lobes, subesophageal, thoracic and abdominal ganglia, as well as in nerve tracts extending through the optic lobes, tritocerebrum, and interganglionic connectives of the pupal stage of these insects. Testis ecdysiotropin is a peptide required by immature moths to initiate production of testes ecdysteroid, which is necessary for the development of the male reproductive system and initiation of spermatogenesis. Antiserum against testis ecdysiotropin also detected an accumulation of testis ecdysiotripic-like material between the inner and outer testis sheaths of pupae. The localization of this peptide in the imaginal disks of the last larval stage, cells and nerve fibers in the optic and antennal lobes of the pupa of both sexes, as well as in the testes during development of the adult reproductive system indicates that testis ecdysiotropin has a much larger impact on adult metamorphosis than development of the reproductive system and initiation of gametogenesis. Although this peptide may have a modulatory role in the central nervous system (CNS), it may also initiate a cascade of activity required for the development of the adult nervous system, in addition to its role in reproduction.

A comparison of three isolation methods for obtaining immunoglobulin A from turkey bile.

Turkey IgA was isolated from bile by three methods: ammonium sulfate precipitation, polyethylene glycol (PEG) extraction, and lambda-carrageenan extraction. The isolated immunoglobulin fractions were compared using double diffusion, immunoelectrophoresis (IE), and enzyme-linked immunosorbent assay (ELISA). Results indicated that all three methods of isolation are sufficient for the initial isolation step for purification of the immunoglobulin fraction in turkey bile. Because of contaminating IgG, IgM, and other high-molecular-weight proteins, further purification by column chromatography is needed to isolate pure IgA. The lambda-carrageenan extraction method appears to be the method of choice for precipitating the immunoglobulin fraction in bile, because of the high antibody activity after extraction. Like ammonium sulfate precipitation, lambda-carrageenan and PEG extraction are not sufficient as single-step purification methods and should be used as the initial step in the purification of IgA.