Gregarina niphandrodes (Eugregarinorida: Septatorina): oocyst surface architecture.

The surface architecture of oocysts produced by Gregarina niphandrodes (Eugregarinorida) from Tenebrio molitor adults (Coleoptera: Tenebrionidae) as revealed by scanning electron microscopy is reported. Gametocysts were allowed to dehisce on 15-mm, round cover glasses; the cover glasses with their oocysts chains were then mounted on stubs without further processing, and sputter-coated with 20-nm gold palladium. Scanning electron microscopy was performed at 10-15 kV with a Hitachi 3000N SEM. Oocysts retained their characteristic shapes as reported in the original species description but showed longitudinal ridges of relatively uniform height, width, and spacing, in separate fields on either side of a central equatorial bulge in the oocysts. There was no ultrastructural evidence of an enclosing external sheath holding the oocysts in a chain. Oocyst ends were flared slightly, and the chain itself was twisted, with adjacent oocysts offset slightly from one another. This article now provides an additional set of structural characters potentially useful in gregarine systematics.

Tumor necrosis factor-alpha induces changes in mitochondrial cellular distribution in motor neurons.

Motor neuron (MN) mitochondrial abnormalities and elevation in spinal fluid levels of the inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) have been implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS). The mechanism of neuron death in ALS remains unclear, along with the contributions of mitochondrial dysfunction and inflammation in the process. Cell cultures enriched for MN derived from embryonic rat spinal cords were established and directly exposed in vitro to recombinant TNF-alpha for varying lengths of time. Although cytokine exposure for up to 4 days failed to induce MN death, mitochondrial changes were observed shortly after initiating treatment. Our results demonstrate that TNF-alpha induced mitochondrial redistribution toward the soma in MN. We postulate that inflammation may precede, and in fact cause, the mitochondrial changes observed in ALS tissue.

New and emended descriptions of gregarines from flour beetles (Tribolium spp. and Palorus subdepressus: Coleoptera, Tenebrionidae).

The following new gregarine taxa are described from larvae of flour beetles (Coleoptera: Tenebrionidae): Awrygregarina billmani, n. gen., n. sp., from Tribolium brevicornis; Gregarina cloptoni, n. sp., from Tribolium freemani; Gregarina confusa, n. sp., from Tribolilum confusum; and Gregarina palori, n. sp., from Palorus subdepressus. In addition, the description of Gregarina minuta Ishii, 1914, from Tribolium castaneum, is emended. Scanning electron micrograph studies of these species' oocysts reveal differences in surface architecture. The Gregarina species have oocysts with longitudinal ridges, visible with SEM, whereas Awrygregarina billmani oocysts have fine circumferential striations; surface architecture is the main feature distinguishing the 2 gregarine genera. Although parasites from adult beetles are not included in the descriptions, adults of all host species can be infected experimentally using oocysts from the new taxa.

Optimization of pheromone lure and trap characteristics for currant clearwing, Synanthedon tipuliformis.

Currant clearwing Synanthedon tipuliformis (Sesiidae) has been a pioneering and successful target of mating disruption in New Zealand, with virtually universal black currant industry adoption since c. 1990. Recent unexplained control failures using mating disruption lead to questions about pheromone efficacy. In this study, we have investigated the possible reasons for reduced control from mating disruption, and report improvements in trap catch based on pheromone loading and trap color. No differences were found in electrophysiological responses to pheromone components from two New Zealand populations. Male moth catches in traps baited with synthetic lures were disrupted in the presence of mating disruption dispensers ( > 99.99%) indicating no apparent barrier to efficacy from the pheromone formulation. Field behavioral observations confirmed this result. Male attraction to yellow delta traps was equivalent to green delta traps, but was greater than to red, black, blue, or white traps. Solid yellow delta traps were more attractive than black traps with yellow stripes, the latter designed to mimic the color pattern of the insect. Solid yellow funnel traps were less attractive than a composite of green, yellow, and white funnel traps. Trap catch increased as a function of pheromone loading, and trap color. In another experiment conducted in Tasmania, there was no difference in catch with single component [(E,Z)-2,13-octadecadienyl acetate] or two component lures [97% (E,Z)-2,13-octadecadienyl acetate:3% (E,Z)-3,13octadecadienyl acetate], refuting the suggestion of a different pheromone strain there.

Virus safety in xenotransplantation: first exploratory in vivo studies in small laboratory animals and non-human primates.

For xenotransplantation, the transplantation of animal cells, tissues and organs into human recipients, to date, pigs are favored as potential donors. Beside ethical, immunological, physiological and technical problems, the microbiological safety of the xenograft has to be guaranteed. It will be possible to eliminate all of the known porcine microorgansims in the nearby future by vaccinating or specified pathogen-free breeding. Thus, the main risk will come from the porcine endogenous retroviruses (PERVs) which are present in the pig genome as proviruses of different subtypes. PERVs will therefore be transmitted, with the xenograft, to the human recipient. PERVs can infect numerous different types of human primary cells and cell lines in vitro and were shown to adapt to these cells by serial passaging on uninfected cells. Furthermore, PERVs have high homology to other retroviruses, such as feline leukemia virus (FeLV) or murine leukemia virus (MuLV), which are known to induce tumors or immunodeficiencies in the infected host. To evaluate the potential risk of a trans-species transmission of PERV in vivo, naive and immunosuppressed rats, guinea pigs and minks were inoculated with PERV and screened over a period of 3 months for an antibody reaction against PERV proteins or for the integration of proviral DNA into the genomic DNA of the host's cells. Furthermore, we inoculated three different species of non-human primates, rhesus monkey (Macaca mulatta), pig-tailed monkey (Macaca nemestrina) and baboon (Papio hamadryas) with high titers of a human-adapted PERV. To simulate a situation in xenotransplantation, the animals received a daily triple immunosuppression using cyclosporine A, methylprednisolone and RAD, a rapamycin derivative, presently under development by Novartis. None of the small laboratory animals or the non-human primates showed production of antibodies against PERV or evidence of integration of proviral DNA in blood cells or cells of several organs, 3 months after virus inoculation, despite the observation that cells of the animals used in the experiment were infectible in vitro. This apparent difference in the outcome of the in vitro and the in vivo data might be explained by an efficient elimination of the virus by the innate or adaptive immunity of the animals.

Mapping full-length porcine endogenous retroviruses in a large white pig.

Xenotransplantation may bridge the widening gap between the shortage of donor organs and the increasing number of patients waiting for transplantation. However, a major safety issue is the potential cross-species transmission of porcine endogenous retroviruses (PERV). This problem could be resolved if it is possible to produce pigs that do not contain replication-competent copies of this virus. In order to determine the feasibility of this, we have determined the number of potentially replication-competent full-length PERV proviruses and obtained data on their integration sites within the porcine genome. We have screened genomic DNA libraries from a Large White pig for potentially intact proviruses. We identified six unique PERV B proviruses that were apparently intact in all three genes, while the majority of isolated proviruses were defective in one or more genes. No intact PERV A proviruses were found in this pig, despite the identification of multiple defective A proviruses. Genotyping of 30 unrelated pigs for these unique proviruses showed a heterogeneous distribution. Two proviruses were uncommon, present in 7 of 30 and 3 of 30 pigs, while three were each present in 24 of 30 pigs, and one was present in 30 of 30 animals examined. Our data indicate that few PERV proviruses in Large White pigs are capable of productive infection and suggest that many could be removed by selective breeding. Further studies are required to determine if all potentially functional proviruses could be removed by breeding or whether gene knockout techniques will be required to remove the residuum.

Analysis of patients treated with living pig tissue for evidence of infection by porcine endogenous retroviruses.

The use of pigs as a source of cells and organs for transplantation has the potential to reduce the current chronic shortage of organs for the treatment of many end-stage diseases. The risk of transmission of infectious agents across the species barrier (zoonoses) has to be assessed. Many such agents can be eliminated from the pig herd. However, porcine endogenous retroviruses, which are carried within the pig genome, are not easily eliminated. They can infect primary and immortalized human cells in vitro, but to date no evidence for in vivo infection has been found in retrospective studies of humans exposed to viable porcine cells. Small-scale clinical trials using porcine cells for the treatment of Parkinson's and Huntington's disease are currently in progress. The prospective monitoring of these patients in conjunction with further research into the biology of this virus will help address safety issues.

The porcine endogenous retrovirus long terminal repeat contains a single nucleotide polymorphism that confers distinct differences in estrogen receptor binding affinity between PERV A and PERV B/C subtypes.

Porcine endogenous retroviruses (PERV) have been shown to have zoonotic potential, both in vitro and in vivo. Once integrated into the host cell genome activation of the proviral genes is ultimately dependent upon transactivation of the long terminal repeat (LTR). Currently there is no direct evidence of host cell transcription factors interacting with PERV LTRs. Using comparative genomics we discovered a potentially functional single nucleotide polymorphism (SNP) within the U5 region downstream of the TATA box in the PERV LTR that distinguishes PERV A from PERV B and PERV C subtypes. We demonstrated that the SNP occurs within a potential hormone-responsive region where it has a profound effect, not only upon estrogen receptor binding but also upon the binding of other transcription factors at this site. These results suggest that differences in transcriptional regulation between PERV subtypes are subtle and, as for other retroviruses, transcription can be mediated by steroid hormone receptors.

Monitoring xenotransplant recipients for infection by PERV.

OBJECTIVES: Concerns have been raised over the possibility of transmission of porcine endogenous retrovirus (PERV) to porcine xenograft recipients. METHODS: To help assess this risk, diagnostic assays capable of detection of an active, latent or cleared PERV infection, and the presence of pig cell microchimerism have been developed by a number of groups. Retrospective studies of patients exposed to living pig tissues have been performed using these assays to look for evidence of cross species transmission. RESULTS: To date no evidence of PERV infection has been found in studies of humans exposed to pig tissues, despite evidence of long lived microchimerism. CONCLUSIONS: These data suggest that PERV infection has not occurred in a clinical setting. However, as infection has been seen in a small animal model further investigation of the risk from PERV is warranted.

In vivo analysis of porcine endogenous retrovirus expression in transgenic pigs.

Xenotransplantation offers a potential solution to the shortage of donor organs for allotransplantation. In vitro studies that demonstrate the transmission of porcine endogenous retroviruses (PERV) from porcine cells to human cells and cell lines have raised concerns regarding the potential transmission of PERV to both xenograft recipients and their contacts (1-4). While no evidence of infection has been detected in any patients who have been treated with a variety of different porcine tissues (5-8), two studies have shown that severe combined immunodeficient (SCID) mice can be infected by PERV after the transplantation of porcine islets (9-10). To further address the concerns of PERV, expression of this virus in tissues and serum from transgenic pigs that express human decay accelerating factor was investigated. Although viral mRNA expression was detected in a variety of tissues, no evidence of viral release was observed in any of the porcine tissues analyzed by transmission electron microscopy. Analysis of porcine serum using the product-based reverse transcriptase assay suggested that virions may be present in porcine serum from large white pigs. However, using methods based on those previously described by Wilson et al. (4), infectious virus was not detected when activated peripheral blood mononuclear cells from these pigs were cocultivated with human cells known to be permissive for PERV.

Fast transport of neurofilament protein along microtubules in squid axoplasm.

Using squid axoplasm as a model system, we have visualized the fast transport of non-filamentous neurofilament protein particles along axonal microtubules. This transport occurs at speeds of 0.5-1.0 microm/second and the majority of neurofilament particles stain with kinesin antibody. These observations demonstrate, for the first time, that fast (0.5-1.0 microm/second) transport of neurofilament proteins occurs along microtubules. In addition, our studies suggest that neurofilament protein can be transported as non-membrane bound, nonfilamentous subunits along axons, and that the transport is kinesin-dependent. Microtubule-based fast transport might therefore provide a mechanism for the distribution and turnover of neurofilament, and perhaps other cytoskeletal proteins, throughout neurons.

Antiretroviral agents inhibit infection of human cells by porcine endogenous retroviruses.

The efficacy of antiretroviral drugs against porcine endogenous retroviruses (PERV) that may be harbored in pig organs intended for transplantation was examined in human cells in vitro. The nucleoside analogs zidovudine and dideoxyinosine were found to effectively inhibit PERV replication.

Sequence and phylogenetic analysis of squid myosin-V: a vesicle motor in nerve cells.

We have shown that vesicles in the axoplasm of the squid giant axon move on actin filaments and that movement is inhibited by myosin V-specific antibodies [Tabb et al., 1998]. In the study reported in this article, experiments were performed to clone and sequence the cDNA for squid brain myosin V. Five proteolytic fragments of purified squid brain myosin V were analyzed by direct protein sequencing [Tabb et al., 1998]. Based on this sequence information, degenerate primers were constructed and used to isolate cDNA clones by PCR. Five clones, representing overlapping segments of the gene, were sequenced. The sequence data and the previous biochemical characterization of the molecule support the classification of this vesicle-associated myosin as a member of the class V myosins. Motif analysis of the head, neck, and tail domains revealed that squid MyoV has consensus sequences for all the motifs found in vertebrate members of the myosin V family of motor proteins. A phylogenetic tree was constructed from a sequence alignment by the neighbor-joining method, using Megalign (DNAStar, Madison, WI); the resulting phylogenetic tree showed that squid MyoV is more closely related to vertebrate MyoV (mouse dilute, chicken dilute, rat myr6, and human myo5a) than Drosophila and yeast (myo2, and myo4) myosins V. These new data on the phylogenetic relationships of squid myosin V to vertebrate myosin V strengthens the argument that myosin V functions as a vesicle motor in vertebrate neurons.

Vesicle transport: the role of actin filaments and myosin motors.

The transport of vesicles and the retention of organelles at specific locations are fundamental processes in cells. Actin filaments and myosin motors have been shown to be required for both of these tasks. Most of the organelles in cells associate with actin filaments and some of the myosin motors required for movement on actin filaments have been identified. Myosin V has been shown to transport endoplasmic reticulum (ER) vesicles in neurons, pigment granules in melanocytes, and the vacuole in yeast. Myosin I has been shown to be involved in the transport of Golgi-derived vesicles in epithelial cells. Myosin VI has been shown to be associated with Golgi-derived vesicles, and cytoplasmic vesicles in living Drosophila embryos. Myosin II may be a vesicle motor but its role in vesicle transport has not been resolved. Secretory vesicles, endosomes and mitochondria appear to be transported on actin filaments but the myosin motors on these organelles have not been identified. Mitochondria in yeast may be transported by the dynamic assembly of an actin "tail." The model that has unified all of these findings is the concept that long-range movement of vesicles occurs on microtubules and short-range movement on actin filaments. The details of how the microtubule-dependent and the actin-dependent motors are coordinated are important questions in the field. There is now strong evidence that two molecular motors, kinesin and myosin V, interact with each other and perhaps function as a complex on vesicles. An understanding of the interrelationship of microtubules and actin filaments and the motors that move cargo on them will ultimately establish how vesicles and organelles are transported to their specific locations in cells.

Search for cross-species transmission of porcine endogenous retrovirus in patients treated with living pig tissue. The XEN 111 Study Group.

Pig organs may offer a solution to the shortage of human donor organs for transplantation, but concerns remain about possible cross-species transmission of porcine endogenous retrovirus (PERV). Samples were collected from 160 patients who had been treated with various living pig tissues up to 12 years earlier. Reverse transcription-polymerase chain reaction (RT-PCR) and protein immunoblot analyses were performed on serum from all 160 patients. No viremia was detected in any patient. Peripheral blood mononuclear cells from 159 of the patients were analyzed by PCR using PERV-specific primers. No PERV infection was detected in any of the patients from whom sufficient DNA was extracted to allow complete PCR analysis (97 percent of the patients). Persistent microchimerism (presence of donor cells in the recipient) was observed in 23 patients for up to 8.5 years.