Investigation of apoptosis in cultured cells infected with equine herpesvirus 1

Many viruses alter different stages of apoptosis of infected cells as a strategy for successful infection. Few studies have addressed mechanisms of equine herpesvirus 1 (EHV-1) strain-induced cell death. We investigated the effect of an abortigenic strain (AR8 strain) on heterologous Madin–Darby bovine kidney cells and homologous equine dermis (ED) cells cell lines. We compared morphologic and biochemical features of early and late apoptosis at different postinfection times. We investigated translocation of phosphatidylserine to the cell surface, nuclear fragmentation and changes in the cytoskeleton using flow cytometry and annexin V/propidium iodide staining, DNA laddering, terminal deoxynucleotidyl transferase UTP nick-end labeling assay and immunofluorescence staining of cytokeratin 18 cleavage. AR8 EVH-1 strain interfered with apoptosis in both cell lines, particularly during the middle stage of the replication cycle; this was more evident in ED cells. Although this antiapoptotic effect has been reported for other alpha herpesviruses, our findings may help elucidate how EHV-1 improves its infectivity during its cycle.     

Determination of synthesis, recycling and body mass of glucose in rats and rabbits in vivo with 3H- and 14C-labelled glucose

1. Glucose labelled with (3)H in position 2 and uniformly with (14)C was administered simultaneously to rabbits and rats either as a single injection or by continuous infusion. Plasma glucose specific radioactivity and the yield of (3)H in the plasma water were monitored. 2. The rates of synthesis, recycling of carbon and total body mass of glucose were calculated, without assuming a multicompartmental model and without fitting data by exponential expressions. 3. The rate of synthesis of glucose in starved-overnight rabbits was 4mg/min per kg (range 3-4.5mg/min per kg) and 25-35% of the glucose carbon was recycled. The mass of total body glucose in starved rabbits was 290mg/kg (range 220-390mg/kg). About one-third of the total body glucose equilibrates nearly instantaneously with plasma glucose. 4. In rats starved overnight, glucose synthesis was about 10mg/min per kg and recycling of carbon ranged from 30-40%. Total body mass (per kg body weight) is similar to that in rabbits. 5. The activity in plasma water after injection of [2-(3)H]glucose was determined. The initial rate of (3)H(2)O formation is rapid, indicating that the major site of glucose catabolism is in the rapidly mixing pool. The curve of total body glucose radioactivity was obtained from the (3)H(2)O yield, and total mass of glucose was calculated. This agrees with that obtained from the (3)H specific-radioactivity curve.     

IL-15 can signal via IL-15Rα, JNK, and NF-κB to drive RANTES production by myeloid cells

IL-15 plays many important roles within the immune system. IL-15 signals in lymphocytes via trans presentation, where accessory cells such as macrophages and dendritic cells present IL-15 bound to IL-15Rα in trans to NK cells and CD8(+) memory T cells expressing IL-15/IL-2Rβ and common γ chain (γ(c)). Previously, we showed that the prophylactic delivery of IL-15 to Rag2(-/-)γ(c)(-/-) mice (mature T, B, and NK cell negative) afforded protection against a lethal HSV-2 challenge and metastasis of B16/F10 melanoma cells. In this study, we demonstrated that in vivo delivery of an adenoviral construct optimized for the secretion of human IL-15 to Rag2(-/-)γ(c)(-/-) mice resulted in significant increases in spleen size and cell number, leading us to hypothesize that IL-15 signals differently in myeloid immune cells compared with lymphocytes, for which IL-15/IL-2Rβ and γ(c) expression are essential. Furthermore, treatment with IL-15 induced RANTES production by Rag2(-/-)γ(c)(-/-) bone marrow cells, but the presence of γ(c) did not increase bone marrow cell sensitivity to IL-15. This IL-15-mediated RANTES production by Rag2(-/-)γ(c)(-/-) bone marrow cells occurred independently of the IL-15/IL-2Rβ and Jak/STAT pathways and instead required IL-15Rα signaling as well as activation of JNK and NF-κB. Importantly, we also showed that the trans presentation of IL-15 by IL-15Rα boosts IL-15-mediated IFN-γ production by NK cells but reduces IL-15-mediated RANTES production by Rag2(-/-)γ(c)(-/-) myeloid bone marrow cells. Our data clearly show that IL-15 signaling in NK cells is different from that of myeloid immune cells. Additional insights into IL-15 biology may lead to novel therapies aimed at bolstering targeted immune responses against cancer and infectious disease.     

Maintenance of quantitative genetic variance in complex,multitrait phenotypes: the contribution of rare,large effect variants in 2 Drosophila species

The interaction of evolutionary processes to determine quantitative genetic variation has implications for contemporary and future phenotypic evolution, as well as for our ability to detect causal genetic variants. While theoretical studies have provided robust predictions to discriminate among competing models, empirical assessment of these has been limited. In particular, theory highlights the importance of pleiotropy in resolving observations of selection and mutation, but empirical investigations have typically been limited to few traits. Here, we applied high-dimensional Bayesian Sparse Factor Genetic modeling to gene expression datasets in 2 species, Drosophila melanogaster and Drosophila serrata, to explore the distributions of genetic variance across high-dimensional phenotypic space. Surprisingly, most of the heritable trait covariation was due to few lines (genotypes) with extreme [>3 interquartile ranges (IQR) from the median] values. Intriguingly, while genotypes extreme for a multivariate factor also tended to have a higher proportion of individual traits that were extreme, we also observed genotypes that were extreme for multivariate factors but not for any individual trait. We observed other consistent differences between heritable multivariate factors with outlier lines vs those factors without extreme values, including differences in gene functions. We use these observations to identify further data required to advance our understanding of the evolutionary dynamics and nature of standing genetic variation for quantitative traits.     

Stent implantation through a self-expanding stent

Jailing of a side-branch is a known complication of stent implantation, and makes access to the side-branch difficult, especially if the stent is of the self-expanding type. Although plain balloon angioplasty is feasible for the jailed side-branches, the use of newer devices (a stent, Rotablation or atherectomy) has not been described. We describe a novel way of treating a side-branch jailed by a self-expanding stent by using stent implantation through the strut of a self-expanding stent.     

Patterns of development of gonads, sex-drive and hormonal responses in tropical beef bulls

The development of different traits was studied in tropical beef bulls of seven genotypes (Brahman, Africander, British and combinations of these) from approximately 500 to 910 d of age. Bulls were raised under pasture conditions without supplementation. At each examination, approximately 2 mo apart, bulls were weighed, palpated (including scrotal and testicular measurement), electroejaculated, and subjected to two libido tests with estrus-induced females. At alternate examinations, plasma luteinizing hormone (LH) was measured at 30 and 150 min post gonadotrophin releasing hormone (GnRH) injection (LH - 30 and LH - 150) and testosterone (T) was measured at 150 min (T - 150). In general, nutritional and environmental stressors appeared to impede bull reproductive development. Scrotal circumference increased nonlinearly, apparently influenced by puberty and average daily gain (ADG). Libido increased overall, albeit nonlinearly also. No apparent marked differences in development of either trait could be attributed to genotype differences, although Brahman bulls tended to display lower sexual interest. The LH-30 level was relatively high (>14 ng/ml) at 500 and 640 d of age, but then dropped markedly at 760 d followed by a slight recovery. The LH-150 level followed a similar pattern, although it was very low at 500 d of age. The T-150 level showed a reverse pattern, being lower initially and higher in the latter part of the study. No apparent genotype differences occurred. Possible contributory influences on these patterns, including the onset of puberty and sexual maturity, season and nutrition, are discussed herein.     

Beef bulls mated to estrus synchronized heifers: Single- vs multi-sire breeding groups

Beef bulls of approximately 15 months of age were placed with heifers at bull to female ratios (BFR) of 1:20 (n = 3) and 2:40 (n = 3) to compare the breeding efficiency of bulls used in either a single- or a multi-sire group. Prior to the breeding period, each bull was given a breeding soundness exam and two exposures to a libido/serving capacity test. For the purpose of synchronizing estrus, heifers received a nine-day Syncro-Mate-B((R)) (G. D. Searle & Co.) treatment. Twenty-seven hr after removal of the implants, bulls were placed with heifers and sexual activity was observed continuously for the succeeding 30 hr. With the exception of number of services per heifer, the mating performance of bulls and pregnancy rates at both BFR's were not different (P>.05). Heifers in single-sire groups were serviced more times (P<.05) than those in multi-sire groups (4.1 +/- 0.6 vs 2.6 +/- 0.2, respectively). Approximately 50% of heifers in multi-sire groups were serviced by both bulls. Due to the overlap in servicing heifers and the non-significant difference in pregnancy rates between the two BFR's, it was concluded that a single-sire mating program was more efficient.     

Relationships among production traits and estimates of sex drive and dominance value in yearling beef bulls

Average daily gain (ADG) and final test weight (FTW) were compared with libido score (L), serving capacity score (SC), reaction time to first mount (RTM), reaction time to first service (RTS) and dominance value (DV) in 90 purebred yearling beef bulls which had just completed an 140-day performance test. The bulls represented 15 breeding lines (12 Hereford, 2 Black Angus and 1 Red Angus) and nine sires within lines. Libido and serving capacity assessments were applied to randomly selected groups of two bulls which were tested for 10-minutes with nonestrous heifers restrained in service crates. RTM and RTS were recorded for both tests. Social dominance was assessed during randomized observations of social interactions between bulls within their pen groups (comprising 9 to 10 bulls) over four days. Two methods of determining social dominance (DV1 and DV2) were employed initially. Differences occurred among both lines and sire/lines in mean and best libido scores (P < .05). Sire/lines differed in L1, while both lines and sire/lines differed in L2 (P < .05). These differences were not reflected in either reaction times or serving capacity scores. Sires and sire/lines both differed in FTW (P < .05 and P < .01 respectively), while DV1 differed with sire/lines (P < .01). DV1 and DV2 were highly correlated (r = .84). Those sex-drive estimates which correlated with DV1 were the mean and best libido scores, mean serving capacity score, L2 and RTS 1 (P < .05). All correlations were negative, indicating that dominance and sex-drive may be unfavourably related in yearling beef bulls. ADG and FTW showed negative correlations with the mean and best serving capacity scores and with SC2 (P < .05). The only positive correlations between the two production traits and a sex-drive estimate was with RTM 2 (P < .05). This indicates that ADG and FTW may be unfavourably related to sex-drive in yearling beef bulls.     

The determination of lactate turnover in vivo with 3H- and 14C-labelled lactate. The significance of sites of tracer administration and sampling.

The D-ribulokinase and D-xylulokinase of Klebsiella aerogenes were purified to homogeneity from Escherichia coli K12 construct strains that synthesized these enzymes constitutively. The D-ribulokinase, which is encoded in the ribitol operon, is active as a dimer of 60 000 subunit mol.wt., whereas the D-xylulokinase, which is encoded in the D-arabitol operon, is active as a dimer of 54 000 subunit mol.wt. The amino acid compositions and N-terminal sequences of both pentulokinases are reported. The Kapp. values of the enzymes for their D-pentulose substrates were determined, and the D-ribulokinase was shown to have a low-affinity side-specificity for ribitol and D-arabitol. These results are discussed in the context of the evolution of the Klebsiella aerogenes pentitol operons.     

Structure and function of human C5a anaphylatoxin. Selective modification of tyrosine 23 alters biological activity but not antigenicity

Reaction of either human C5a or its des-Arg74 derivative (des-Arg74-C5a) with tetranitromethane under nondenaturing conditions results in selective nitration of only 1 of the 2 tyrosine residues found in these glycopolypeptides. This reactive tyrosyl residue was identified as that found in position 23 of the sequence. Nitrotyrosyl23-C5a and -des-Arg74-C5a were separated from their respective unmodified precursors by cation-exchange fast protein liquid chromatography. These purified derivatives served as reagents for the subsequent preparation of both aminotyrosyl23-C5a and -des-Arg74-C5a as well as photoreactive analogs of C5a. Radioimmunoassays performed with C5a derivatives serving as competing ligands and a murine antihuman C5a monoclonal antibody employed as first antibody demonstrated that selective modification of tyrosine23 did not produce a detectible alteration in the antigenic properties of C5a. By contrast, either nitro- or aminotyrosyl23-C5a was approximately 3-fold less active than native C5a in both bioassays and competitive ligand-receptor binding assays. Additionally, photoreactive derivatives prepared by coupling either N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)-hexanoate or p-nitrophenyl-2-diazo-3,3,3-trifluoropropionate to aminotyrosyl23-C5a at pH 5.0 failed to interact with the neutrophil C5a receptor. These observations suggest that the tyrosyl23 residue of C5a may participate importantly in the binding interactions of this chemotactic factor and its granulocyte receptor.