The effect of feeding tall fescue seed infected by Acremonium coenophialum on pregnancy and parturition in female rats

1. Female Sprague-Dawley rats (Rattus norvegicus) were randomly assigned to various dietary treatments containing: (1) 100% Purina rodent chow, ad libitum; (2) same as 1, but restricted to daily intake of 7; (3) 50% rodent chow (w/w) and 50% endophyte-free tall fescue (Festuca arundinacea) seed; (4) same as 3, but restricted to intake of 5; (5) 50% rodent chow, 25% endophyte-free tall fescue seed and 25% endophyte-infected (Acremonium coenophialum) tall fescue seed; (6) 50% rodent chow, 12.5% endophyte-free and 37.5% endophyte-infected tall fescue seed; and (7) 50% rodent chow and 50% endophyte infected tall fescue seed. 2. Average daily feed intakes and average daily weight gains decreased with higher levels of endophyte infected seed. 3. Frequency of litter production was affected by all endophyte-infected containing diets. 4. Conception was reduced only in dietary treatment (7). 5. Litter weights, number of pups per litter and weight per pup were proportionally reduced as higher levels of infected seed were incorporated in the ingested diets.     

Failure of embryos from bluetongue infected cattle to transmit virus to susceptible recipients or their offspring

Sixty heifers were infected with bluetongue virus (BTV) by the bites of the vector and by inoculation with insect origin virus. During the acute and convalescent stages of the infection, embryos were collected nonsurgically from these animals and washed according to the recommendations of the International Embryo Transfer Society (1). No BTV was isolated from 77 of these embryos when they were inoculated onto cell culture and into embryonating chicken eggs. There was no evidence of lateral BTV transmission when 231 of these embryos were transferred into susceptible recipients, nor was there evidence of vertical BTV transmission to the 88 calves resulting from these transfers. Another six donors that were assumed to have recovered from a natural infection of BTV, were added to the study to increase the probability of obtaining embryos from a persistently infected BTV carrier. However, it was determined later that these animals had not been infected with BTV but with the closely-related epizootic hemorrhagic disease virus (EHDV). Embryos were collected from these donors and washed as above. Neither BTV nor EHDV was isolated from 26 of these embryos by the inoculation of cell culture and embryonating chicken eggs. There was no evidence of lateral BTV or EHDV transmission to recipients of 15 of these embryos or of vertical BTV or EHDV transmission to the resulting 7 calves. However, two recipients of embryos from one of these donors developed antibodies to BTV 6 to 9 months after transfer. Passive antibodies to BTV were also detected in their calves. There is good evidence that these two recipients acquired BTV from natural exposure to infected insect vectors and not from the transferred embryos.     

SB subregion of the human major histocompatibility complex: gene organization, allelic polymorphism and expression in transformed cells

The SB region of the human major histocompatibility complex (MHC) has been cloned from cosmid and lambda phage libraries made from the human B-lymphoblastoid cell line Priess (DR4/4, DC4/4, SB3/4). Two alpha genes and two beta genes are encoded in the 100 kb long SB region in the order SB alpha-SB beta-SX alpha-SX beta. The SB alpha and SB beta genes encode the alpha and beta subunits of the SB subset of class II MHC molecules. Both the SX alpha and the SX beta genes are pseudogenes in the haplotype examined. From the isolated clones, the two haplotypes of the Priess cell line, SB3 and SB4, are distinguished by nucleotide sequencing and blot hybridization analyses. Restriction site polymorphisms between the SB3 and SB4 clones were observed only in relatively small regions of the SB beta and SX beta genes. A mouse macrophage cell line was transfected with one of the cosmid clones containing both SB alpha and SB beta genes. Expression of the alpha and beta genes was detected by fluorescene-activated cell sorting (FACS) and two-dimensional gel electrophoresis using SB-specific monoclonal antibodies.     

Inhibition of human and simian immunodeficiency virus protease function by targeting Vpx-protease-mutant fusion protein into viral particles.

The human immunodeficiency virus type I (HIV-1) Vpr and HIV-2 Vpx proteins package into virions through interactions with their cognate Gag polyprotein precursor. The targeting properties of Vpr and Vpx have been exploited to incorporate foreign proteins into virions by expression as heterologous fusion molecules (X. Wu, H.-M. Liu, H. Xiao, J. Kim, P. Seshaiah, G. Natsoulis, J. D. Boeke, B. H. Hahn, and J. C. Kappes, J. Virol. 69:3389-3398, 1995). To explore the possibility of utilizing Vpx and Vpr to target dominant negative mutants of the HIV Pol proteins into virions, we fused HIV-2 Vpx with an enzymatically defective protease (PR) mutant. Using a vector system to facilitate transient coexpression with HIV provirus, Vpx-PR-mutant (VpxPR(M)) fusion protein was expressed and packaged efficiently into HIV-2 and simian immunodeficiency virus virions. Immunoblot analysis of purified virions demonstrated that the packaging of VpxPR(M) interfered with the processing of the Gag and Gag/Pol precursor proteins, similar to that of a well-characterized active-site PR inhibitor. The incomplete processing of Gag and Gag/Pol was consistent with a 25-fold reduction in virion infectivity. The coexpression of a packaging defective VpxPR(M) fusion protein with HIV-2 provirus produced virions with fully processed Gag protein, similar to wild-type virions. Importantly, virions trans complemented with a Vpx-chloramphenicol acetyltransferase fusion protein were normal with respect to the processing of Gag protein and the ability to infect and replicate in vitro. These results indicate that VpxPR(M) specifically inhibited the function of the viral protease and provide for the first time proof of principle that the incorporation of foreign proteins into virions via fusion with Vpx can inhibit HIV replication. The use of accessory proteins as vehicles to deliver deleterious proteins to virions, including dominant negative mutants of Pol proteins, may provide new opportunities for application of gene therapy-based antiretroviral strategies. The ability to package PR by expression in trans, independent of the Gag/Pol precursor, also represents a novel approach that may be exploited to study the function of the Pol proteins.     

L cells expressing DQ molecules of the DR3 and DR4 haplotypes: reactivity patterns with mAbs

cDNAs coding for the HLA class II DR and DQ and chains of the diabetogenic haplotypes DR3 and DR4 were introduced into a mammalian expression vector and transfected into L-cell mouse fibroblasts to produce cells expressing individual human class II molecules. Stable L transfectants were generated expressing each of the DR or DQ isotypes of the cis-encoded and chains of the DR3 or DR4 haplotypes, as well as the trans-encoded and chains of the DQ molecules of the two haplotypes. However, isotype mismatched combinations (DR/DQ or DQ/DR) did not result in any stable transfectants. The stable DQ L-cell transfectants obtained, along with homozygous B-cell lines expressing the DQ2 and DQ8 specificities, were tested against a large panel of twentyone anti-HLA class II monoclonal antibodies (mAbs). Their unusual reactivity patterns are described including the failure of most pan-DQ mAbs to react with all DQ expressing L-cell transfectants. Interestingly, some mAbs react with certain heterodimers expressed on B-LCL but fail to recognize the same heterodimers expressed on the transfectants. This is suggestive of minor structural modifications that class II molecules undergo depending on the cells they are expressed on.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number U07848. The name DQB1 ^* 0202 was officially assigned by the WHO Nomenclature Committee in April 1994. This follows the agreed policy that, subject to the conditions stated in the most recent Nomenclature Report (Bodmer et al. 1992), names will be assigned to new sequences as they are identified. Lists of such new names will be published in the following WHO Nomenclature Report     

Targeting foreign proteins to human immunodeficiency virus particles via fusion with Vpr and Vpx.

The human immunodeficiency virus type 1 (HIV-1) and HIV-2 Vpr and Vpx proteins are packaged into virions through virus type-specific interactions with the Gag polyprotein precursor. To examine whether HIV-1 Vpr (Vpr1) and HIV-2 Vpx (Vpx2) could be used to target foreign proteins to the HIV particle, their open reading frames were fused in frame with genes encoding the bacterial staphylococcal nuclease (SN), an enzymatically inactive mutant of SN (SN*), and chloramphenicol acetyltransferase (CAT). Transient expression in a T7-based vaccinia virus system demonstrated the synthesis of appropriately sized Vpr1-SN/SN* and Vpx2-SN/SN* fusion proteins which, when coexpressed with their cognate p55Gag protein, were efficiently incorporated into virus-like particles. Packaging of the fusion proteins was dependent on virus type-specific determinants, as previously seen with wild-type Vpr and Vpx proteins. Particle-associated Vpr1-SN and Vpx2-SN fusion proteins were enzymatically active, as determined by in vitro digestion of lambda phage DNA. To determine whether functional Vpr1 and Vpx2 fusion proteins could be targeted to HIV particles, the gene fusions were cloned into an HIV-2 long terminal repeat/Rev response element-regulated expression vector and cotransfected with wild-type HIV-1 and HIV-2 proviruses. Western blot (immunoblot) analysis of sucrose gradient-purified virions revealed that both Vpr1 and Vpx2 fusion proteins were efficiently packaged regardless of whether SN, SN*, or CAT was used as the C-terminal fusion partner. Moreover, the fusion proteins remained enzymatically active and were packaged in the presence of wild-type Vpr and Vpx proteins. Interestingly, virions also contained smaller proteins that reacted with antibodies specific for the accessory proteins as well as SN and CAT fusion partners. Since similar proteins were absent from Gag-derived virus-like particles and from virions propagated in the presence of an HIV protease inhibitor, they must represent cleavage products produced by the viral protease. Taken together, these results demonstrate that Vpr and Vpx can be used to target functional proteins, including potentially deleterious enzymes, to the human or simian immunodeficiency virus particle. These properties may be exploitable for studies of HIV particle assembly and maturation and for the development of novel antiviral strategies.     

Anchoring of bovine chromosomes 4, 6, 7, 10, and 14 linkage group telomeric ends via FISH analysis of lambda clones

We report the placement of 34 new microsatellite (ms) markers, isolated from a lambda phage genomic clone library, on the bovine genetic map by linkage to published markers. Five of these markers lie at or near the ends of linkage groups and are used to establish chromosomal coverage and orientation. Fluorescence in situ hybridization (FISH) analysis demonstrates that the linkage groups on the U.S. Meat Animal Research Center (MARC) map extend to the telomeric region of Chromosomes (Chrs) 7 and 10. Linkage groups on Chrs 4, 6, and 14 appear to be less inclusive. Received: 23 September 1996 / Accepted: 28 December 1996     

Species- and clone-specific responses to environmental stimuli in the cladocerans Bosmina cornuta and B. pellucida - a comparison with Daphnia

We studied the variation of small-scale swimming behaviour in eight Bosmina cornuta and ten B. pellucida clones in response to key environmental factors to test whether swimming behaviour and genotypes are linked in non-Daphnia cladocerans. We quantified (1) the short-term responses to changes in temperature, light intensity and pH, (2) the response to long-term temperature acclimation, and (3) the pH-related survival rates. Vertical swimming activity S was quantified in cuvette experiments as crossings of a line at 2 cm height per individual an hour. S differed significantly among species and conspecific clones. At any temperature, light intensity and pH tested, B. cornuta (clone variation: 40-58 crossings/ind.- h) showed a higher vertical swimming activity than B. pellucida (clone variation: 25-48 crossings/ind.- h). A short-term change of water temperature (range tested: 10-25C) only affected S of B. cornuta, whereas that of B. pellucida remained unaltered. In contrast, S increased with rising temperature following long-term temperature acclimation (range tested: 10-20C) in both species. Swimming activity was inversely related to the light intensity (range tested: 60-60,000 lux), but decrease of activity was stronger in B. pellucida (44′ 12 crossings/ind - h) than in B. cornuta (50′ 40 crossings/ind.- h). Short-term changes of pH (range tested: 4-6) did not influence swimming activity in any species, although a prolonged exposure (24 h) to pH 4 was lethal. Thus, Bosmina showed behavioural responses which permit to distinguish between the species and which are related to their seasonal succession and distribution pattern.