Papaverine-sensitive phosphodiesterase activity is measured in bovine spermatozoa.

Cyclic adenosine monophosphate (cAMP) plays a crucial role as a signaling molecule for capacitation, motility, and acrosome reaction in mammalian spermatozoa. It is well-known that cAMP degradation by phosphodiesterase (PDE) enzyme has a major impact on sperm functions. This study was undertaken to characterize cAMP-PDE activity in bovine spermatozoa. Total cAMP-PDE activity in cauda epididymal and ejaculated spermatozoa was 543.2 ± 49.5 and 1252.6 ± 86.5 fmoles/min/106 spermatozoa, respectively. Using different family-specific PDE inhibitors, we showed that in cauda epididymal and ejaculated spermatozoa, the major cAMP-PDE activity was papaverine-sensitive (44.5% and 57.5%, respectively, at 400 nm, papaverine is a specific inhibitor of the PDE10 family). These data are supporting the functional presence of PDE10 in bovine spermatozoa and were further confirmed by western blot to be PDE10A. Using immunocytochemistry, we showed immunoreactive signal for PDE10A present on the post-acrosomal region of the head and on the flagella of ejaculated spermatozoa. Using papaverine, we showed that it promotes tyrosine phosphorylation of sperm proteins, phosphorylation of Erk1 and Erk2, and Ca2+ release from Ca2+ store. These results suggest that PDE10 is functionally present in bovine spermatozoa and is affecting different molecular events involved in capacitation, most probably by cAMP local regulation.

Low temperature induced defence gene expression in winter wheat in relation to resistance to snow moulds and other wheat diseases.

Cold hardening of winter wheat at 2 °C for 1-6 wks increased resistance to the snow mould pathogens LTB, Typhula incarnata, and Microdochium nivale as well as to powdery mildew (Blumaria graminis f. sp. graminis) and stripe rust (Puccinia striiformis). Using microarrays and hardening of winter wheat for 0.25, 0.5, 1, 7, 21 and 49 d, an upregulation of a wide range of stress-response genes that include defence-related and abiotic stress-related genes, transcription factors including several lipoxygenases and ethylene responsive factors, and WRKY genes was observed. For the majority of these genes, the upregulation occurred later in the 21-49 d hardening treatments and coincided with the highest expression levels of snow mould resistance. Defence-related sequences were upregulated to a greater extent and were more numerous in the snow mould resistant line CI14106 compared to cold hardy DH+268. Transcript profiling of candidate defence and other stress-related genes under prolonged conditions at -3 °C with or without snow mould infection showed that there was a decline in transcripts of the defence-related genes PR1.1b and NPR3 during the 12wks incubation. Additionally, 14 d hardening was insufficient to permit full expression of the jasmonic acid synthesis gene, allene oxide synthase (AOS) and the fructan degrading enzyme ß-fructofuranosidase compared the 42 d hardening treatment. The snow mould resistant line CI14106 was able to maintain higher transcript levels of AOS for longer conditions compared to the susceptible line Norstar under artificial snow mould conditions. These results explain the nature of cold-induced resistance to snow moulds and provide direction on establishing selection criteria for improving resistance and cold tolerance in winter wheat.

Clinical outcomes of first antiretroviral regimen in HIV/hepatitis C virus co-infection.

OBJECTIVES: Despite the benefits of HAART, initiation of antiretroviral therapy in HIV-HCV co-infected patients is often delayed as a consequence of patient and physician concern pertaining to liver toxicity. It is unclear whether this is justified. METHODS: We retrospectively evaluated treatment duration and outcome in 186 patients initiating a first HAART regimen. RESULTS: Despite frequent HIV RNA suppression and CD4 T-cell increase following initiation of HAART, the median duration of therapy was only 8 months. Therapy was discontinued primarily for gastrointestinal intolerance (26%), poor adherence (19%), neurocognitive side effects (13%), and substance abuse (6%). Regimes were changed to reduce pill burden and/or frequency of dosing as well (11%). Only six (4%) subjects interrupted therapy as a result of clinically apparent liver toxicity. None were on low dose ritonavir-containing therapy. In those subjects remaining on HAART for at least 12 months, the median ALT level increased marginally from a baseline of 44 IU/mL to 56 IU/mL. The median AST was 44 IU/mL at baseline and at month 12. CONCLUSIONS: These results support our contention that regimen potency, durability, and extrahepatic side effect profile should remain the paramount considerations related to the selection of HAART regimen in HIV-HCV co-infection.

Inheritance and QTL analysis of durable resistance to stripe and leaf rusts in an Australian cultivar, Triticum aestivum 'Cook'.

An F4-derived F6 recombinant inbred line population (n = 148) of a cross between the durable stripe (yellow) rust (caused by Puccinia striiformis) and leaf (brown) rust (caused by Puccinia triticina) resistant cultivar, Triticum aestivum 'Cook', and susceptible genotype Avocet-YrA was phenotyped at several locations in Canada and Mexico under artificial epidemics of leaf or stripe rusts and genotyped using amplified fragment length polymorphism (AFLP) and microsatellite markers. Durable adult plant resistance to stripe and leaf rusts in 'Cook' is inherited quantitatively and was based on the additive interaction of linked and (or) pleiotropic slow-rusting genes Lr34 and Yr18 and the temperature-sensitive stripe rust resistance gene, YrCK, with additional genetic factors. Identified QTLs accounted for 18% to 31% of the phenotypic variation in leaf and stripe rust reactions, respectively. In accordance with the high phenotypic associations between leaf and stripe rust resistance, some of the identified QTLs appeared to be linked and (or) pleiotropic for both rusts across tests. Although a QTL was identified on chromosome 7D with significant effects on both rusts at some testing locations, it was not possible to refine the location of Lr34 or Yr18 because of the scarcity of markers in this region. The temperature-sensitive stripe rust resistance response, conditioned by the YrCK gene, significantly contributed to overall resistance to both rusts, indicating that this gene also had pleiotropic effects.

Molecular cytogenetic discrimination and reaction to wheat streak mosaic virus and the wheat curl mite in Zhong series of wheat--Thinopyrum intermedium partial amphiploids.

Thinopyrum intermedium (2n = 6x = 42, JJJsJsSS) is potentially a useful source of resistance to wheat streak mosaic virus (WSMV) and its vector, the wheat curl mite (WCM). Five partial amphiploids, namely Zhong 1, Zhong 2, Zhong 3, Zhong 4, and Zhong 5, derived from Triticum aestivum x Thinopyrum intermedium crosses produced in China, were screened for WSMV and WCM resistance. Zhong 1 and Zhong 2 had high levels of resistance to WSMV and WCM. The other three partial amphiploids, Zhong 3, 4, and 5, were resistant to WSMV, but were susceptible to WCM. Genomic in situ hybridization (GISH) using a genomic DNA probe from Pseudoroegneria strigosa (SS, 2n = 14) demonstrated that two partial amphiploids, Zhong 1 and Zhong 2, have almost the identical 10 Th. intermedium chromosomes, including four Js, four J, and two S genome chromosomes. Both of them carry two pairs of J and a pair of Js genome chromosomes and two different translocations that were not observed in the other three Zhong lines. The partial amphiploids Zhong 3, 4, and 5 have another type of basic genomic composition, which is similar to a reconstituted alien genome consisting of four S and four Js genome chromosomes of Th. intermedium (Zhong 5 has two Js chromosomes plus two Js-W translocations) with six translocated chromosomes between S and Js or J genomes. All three lines carry a specific S-S-Js translocated chromosome, which might confer resistance to barley yellow dwarf virus (BYDV-PAV). The present study identified a specific Js2 chromosome present in all five of the Zhong lines, confirming that a Js chromosome carries WSMV resistance. Resistance to WCM may be linked with J or Js chromosomes. The discovery of high levels of resistance to both WSMV and WCM in Zhong 1 and Zhong 2 offers a useful source of resistance to both the virus and its vector for wheat breeding programs.

Different Reactions to the Wheat Curl Mite and Wheat streak mosaic virus in Various Wheat-Haynaldia villosa 6V and 6VS Lines.

Wheat curl mite (WCM), Aceria tosichella, is the vector of Wheat streak mosaic virus (WSMV), a destructive viral pathogen in wheat (Triticum aestivum). Genetic resistance to WCM colonization can reduce the incidence of wheat streak mosaic. Chromosome 6V in Hay-naldia villosa is a new source of WCM resistance. We compared variation in resistance among different sources of H. villosa chromosome 6V and 6VS lines to WCM and WSMV and their effectiveness in controlling the incidence of WSMV following exposure to viruliferous WCM. WCM resistance varied among the 6V and 6VS lines depending on the H. villosa parent. The 6V substitution lines Yi80928, GN21, and GN22 derived from an accession of H. villosa from China, and the 6VS translocation lines 92R137, 92R178, and Sub6V from an H. villosa accession collected from the United Kingdom were uniformly resistant to WCM colonization. In contrast, the 6V substitution line RW15 and a 6VS translocation line Pm33 developed from an H. villosa collection from the former Union of Soviet Socialist Republics were susceptible to WCM. All 6V and 6VS lines were susceptible to WSMV when manually inoculated. However, symptom expression was delayed in the WCM-resistant 6V and 6VS lines after exposure to viruliferous WCM. The 6V and 6VS lines differed in their ability to control WSMV infection. WCM-susceptible lines RW15 and Pm33 had no effect on controlling the infection by WSMV. Lines GN21 and GN22 were the most effective of the three H. villosa sources in limiting the spread of WSMV. Their high yield potential and protein content, in combination with resistance to stripe rust (Puccinia striiformis f. sp. tritici) and powdery mildew (Erysiphe graminis f. sp. tritici), make GN21 and GN22 promising sources of WCM resistance.

Characterisation of Triticum vavilovii-derived stripe rust resistance using genetic, cytogenetic and molecular analyses and its marker-assisted selection.

Stripe rust resistance was identified in Triticum vavilovii( T. vaviloviiAus22498)-derived Russian wheat aphid (RWA)-resistant germplasm. Inheritance studies indicated monogenic control of resistance. The resistance gene was tentatively designated as Yrvav and was located on chromosome 1B by monosomic analysis. A close association (1.5+/-0.9% recombination) of Yrvav with a T. vavilovii-derived gliadin allele ( Gli-B1vav) placed it in chromosome arm 1BS. Yrvavwas allelic with Yr10. Tests with Yr10 avirulent and virulent pathotypes showed that Yrvav and Yr10 possess identical pathogenic specificity. Yrvav and Yr10 showed close genetic associations with alternate alleles at the Xpsp3000(microsatellite marker), Gli-B1 and Rg1 loci. Based on these observations Yrvav was named as Yr10vav. The close association between Xpsp3000 and Gli-B1 was also confirmed. The Yr10vav-linked Xpsp3000 allele (285 bp) was not present in 65 Australian cultivars, whereas seven Australian wheats lacking Yr10 carried the same Xpsp3000 allele (260 bp) as Yr10carrying wheat cultivar Moro. Xpsp3000 and/or Gli-B1 could be used in marker-assisted selection for pyramiding Yr10vavor Yr10 with other stripe rust resistance genes. Yr10vav was inherited independently of the T. vavilovii-derived RWA resistance.

3(2H)-Benzofuranones and chromanes from liquid cultures of the mycoparasitic fungus Coniothyrium minitans.

Two 3(2H)-benzofuranones and three chromanes were isolated from the mycoparasitic fungus Coniothyrium minitans. Their structures and absolute stereochemistry were determined by spectroscopic methods.

Characterization of microsomal diacylglycerol acyltransferase activity from bovine adipose and muscle tissue.

The activity of the triacylglycerol bioassembly enzyme, diacylglycerol acyltransferase (DGAT), was characterized in microsomal fractions prepared from bovine subcutaneous (SC) adipose, intramuscular (IM) adipose, and muscle (pars costalis diaphragmatis) tissue. The activity of DGAT was generally higher from SC adipose tissue than from IM adipose or muscle tissue. The characteristics of DGAT activity from the three bovine tissues resembled the activity characteristics observed in previous studies from various other organisms and tissues; the pH optimum was near neutrality, the activity was almost completely inhibited by pre-incubation with N-ethylmaleimide (NEM), and the enzyme accepted a broad range of acyl-CoAs and sn-1,2-diacylglycerols. In some aspects, the SC adipose tissue DGAT activity was different from the DGAT activity from the other two tissues. The SC adipose tissue DGAT activity was not as susceptible to inhibition by NEM as the enzymes from the two other tissue sources, and it exhibited increased specificity for substrates containing oleoyl moieties. The differences in DGAT properties between the three bovine tissues may account to some extent for the differences in the relative fatty acid composition and the positional distribution of fatty acids in triacylglycerol between bovine tissues. The observed differences in enzymatic properties also support recent biochemical and molecular genetic observations that imply the existence of multiple DGAT genes and/or isoforms.