Artificial insemination technology for the emu--improving sperm survival

For the emu, where monogamous mating is normal, artificial insemination (AI) promises much faster genetic improvement and a considerable reduction in production costs by reducing the number of male birds needed for mating. Semen collection is now a routine procedure so the next step is to develop successful protocols for sperm storage. In this paper, we briefly overview our recent progress on the development of protocols for liquid storage and cryopreservation of emu spermatozoa. We have shown that emu semen can be stored at 10 °C for up to 48 h with a minimal loss of viability, and that cryopreservation with dimethylacetamide (DMA) as a cryoprotectant is feasible because we have observed no adverse effects of this cryoprotectant on the emu sperm membrane integrity, morphology and motility. We now need to establish the predictability of the various tests in vivo, but the proportions of live normal and motile sperm with good egg membrane penetration potential suggest that acceptable numbers of competent sperm are preserved and that this will be sufficient for AI.     

The effect of temperature and pH on the motility and viability of ostrich sperm

As the chemical environment of semen diluents can have a profound effect on sperm quality, we examined the effect of temperature and pH on the motility and viability of sperm in the ostrich. Semen was collected from four males, each male being replicated three times. Ejaculates were diluted and incubated for 10 min at 20°C and 40°C in four different buffers, temperature adjusted at pH 6, 7, 8 and 9 respectively. Average path velocity (VAP), curvilinear velocity (VCL), straight-line velocity (VSL), linearity (LIN), beat cross frequency (BCF) and amplitude of lateral displacement (ALH) were then recorded for each sample using CASA. The viability of sperm was assessed using nigrosin-eosin staining. Sperm incubated at 40°C had higher motility parameters, except for ALH. At 40°C, VAP, VSL and LIN increased with pH while VCL, BCF and ALH were higher for lower pHs. The viability of sperm was not affected by temperature but decreased at pH values>7. A pH in the neutral range appeared to yield higher quality sperm after in vitro storage at 20°C. However, the effect of different pH levels and temperatures on sperm longevity needs to be investigated further to develop viable ostrich specific diluents.     

Sperm viability, motility and morphology in emus (Dromaius novaehollandiae) are independent of the ambient collection temperature but are influenced by storage temperature

A protocol for storage of emu semen >6 h has not yet been optimized. The objective was to determine: a) whether sperm quality was adversely affected by sudden exposure to low temperatures (5, 10 and 20 °C) during collection; and b) the effects of three storage temperatures (5, 10 and 20 °C) on survival of emu sperm. In two experiments, each repeated three times on alternate days, ejaculates were diluted 1:1 with precooled (5, 10, or 20°C) UWA-E3 diluent and stored for up to 48 h. Collection temperature, or interaction with either the storage time or storage temperature, had no significant effect on sperm viability, motility, or morphology. Mass Motility Score (2.91-3.27 ± 0.26, mean ± SEM), and percentages of live (72.4-76.2 ± 2.4) and morphologically normal sperm (63.3-64.5 ± 2.3) were comparable among collection temperatures. Conversely, storage temperature and storage time affected (P < 0.05) sperm viability, motility, and morphology. After storage for 48 h, percentages of viable, normal, and motile sperm were higher (P < 0.001) at 5 °C (58.7% ± 1.1, 44.7% ± 1.3, and 50.7% ± 4.9, respectively) and 10 °C (62.6% ± 1.1, 54.1% ± 1.3, and 60.4% ± 4.9) than at 20 °C (27.6% ± 1.1, 20.1% ± 1.3, and 25.9% ± 4.9). Beyond 6 h of storage, the percentage of abnormal sperm was higher (P < 0.001) for storage at 5 °C compared to 10 and 20 °C. After 48 h, bacterial counts were considerably higher at 20 °C compared to 5 and 10 °C (P < 0.001). The pH of stored sperm suspension remained unaffected at 5 and 10 °C, but at 20 °C declined to 6.5 ± 0.03 after 24 h (P < 0.05) and to 6.0 ± 0.03 after 48 h (P < 0.001). We concluded that emu semen could be collected at low ambient temperatures (5-20 °C) without compromising its in vitro storage duration and that semen quality during storage for 48 h was better if it was stored at 10 °C than at 5 or 20 °C.     

Identification and Characterization of a Novel Evolutionarily Conserved Lysine-specific Methyltransferase Targeting Eukaryotic Translation Elongation Factor 2 (eEF2)

The components of the cellular protein translation machinery, such as ribosomal proteins and translation factors, are subject to numerous post-translational modifications. In particular, this group of proteins is frequently methylated. However, for the majority of these methylations, the responsible methyltransferases (MTases) remain unknown. The human FAM86A (family with sequence similarity 86) protein belongs to a recently identified family of protein MTases, and we here show that FAM86A catalyzes the trimethylation of eukaryotic elongation factor 2 (eEF2) on Lys-525. Moreover, we demonstrate that the Saccharomyces cerevisiae MTase Yjr129c, which displays sequence homology to FAM86A, is a functional FAM86A orthologue, modifying the corresponding residue (Lys-509) in yeast eEF2, both in vitro and in vivo. Finally, Yjr129c-deficient yeast cells displayed phenotypes related to eEF2 function (i.e. increased frameshifting during protein translation and hypersensitivity toward the eEF2-specific drug sordarin). In summary, the present study establishes the function of the previously uncharacterized MTases FAM86A and Yjr129c, demonstrating that these enzymes introduce a functionally important lysine methylation in eEF2. Based on the previous naming of similar enzymes, we have redubbed FAM86A and Yjr129c as eEF2-KMT and Efm3, respectively.     

Capsule anchoring in Bacillus anthracis occurs by a transpeptidation reaction that is inhibited by capsidin

Bacillus anthracis , the causative agent of anthrax, is a dangerous biological weapon, as spores derived from drug-resistant strains cause infections for which antibiotic therapy is no longer effective. We sought to develop an anti-infective therapy for anthrax and targeted CapD, an enzyme that cleaves poly-γ- d -glutamate capsule and generates amide bonds with peptidoglycan cross-bridges to deposit capsular material into the envelope of B. anthracis . In agreement with the model that capsule confers protection from phagocytic clearance, B. anthracis capD variants failed to deposit capsule into the envelope and displayed defects in anthrax pathogenesis. By screening chemical libraries, we identified the CapD inhibitor capsidin, 4-[(4-bromophenyl)thio]-3-(diacetylamino)benzoic acid), which covalently modifies the active-site threonine of the transpeptidase. Capsidin treatment blocked capsular assembly by B. anthracis and enabled phagocytic killing of non-encapsulated vegetative forms.     

The lipophilic iron compound TMH-ferrocene [(3,5,5-trimethylhexanoyl)ferrocene] increases iron concentrations,neuronal L-ferritin,and heme oxygenase in brains of BALB/c mice

Mismanagement of intracellular iron is a key pathological feature of many neurodegenerative diseases. Our long-term goal is to use animal models to investigate the mechanisms of iron neurotoxicity and its relationship to neurodegenerative pathologies. The immediate aim of this experiment was to determine regional distribution of iron and cellular distribution of iron storage proteins (l- and h-ferritin) and an oxidative stress marker (heme oxygenase-1) in brains of mice fed the lipophilic iron compound (3,5,5-trimethylhexanoyl) (TMH)-ferrocene. We fed male and female weanling BALB/cj mice diets either deficient in iron (0 mg Fe/kg diet), adequate in iron (35 mg Fe/kg diet; control mice), or adequate in iron and supplemented with 0.1 or 1.0 g TMH-ferrocene/kg diet for 8 wk. Iron concentrations in cerebrum were higher in mice fed 1.0 g TMH-ferrocene/kg diet than in control mice (p<0.05). Liver iron concentrations were eightfold higher in mice fed 1.0 g TMH-ferrocene/kg diet than in control mice (p<0.0001). l-Ferritin and heme oxygenase-1 expression were elevated in striatum in mice fed 1.0 g TMH-ferrocene/kg diet. We conculde that administration of the lipophilic iron compound TMH-ferrocene leads to subtle perturbations of cellular iron within the brain, potentially representing a model of iron accumulation similar to that seen in various neuropathological conditions.     

Induction of pathogen resistance and pathogenesis-related genes in tobacco by a heat-stable Trichoderma mycelial extract and plant signal messengers

Heat-stable mycelial extracts of the nonpathogenic fungus Trichoderma longibrachiatum induced resistance in tobacco seedlings ( Nicotiana tabacum L. cv. Wisconsin 38) to the pathogen Phytophthora parasitica var. nicotianae (race 0), which did not involve a hypersensitive response. Resistance could not be induced with mycelial extract prepared in the same manner from P. parasitica . The nonpathogenic mycelial extract induced expression of PR-1b and osmotin (PR-5) genes to a higher level than did mycelial extract from the pathogenic fungus. The tissue-specific pattern of PR gene induction by the nonpathogenic mycelial extract was different from that of the pathogenic mycelial extract and was consistent with the ability of the former to cause disease resistance. The expression patterns of these two PR genes and the accumulations of their encoded proteins also were affected by salicylic acid (SA), methyl jasmonate (MeJA), ethylene (E) and combinations of these plant signal messengers. However, only combined SA and MeJA treatment mimicked the pattern of PR gene mRNA and protein accumulation induced by the nonpathogenic mycelial extract. E inhibitors blocked both mycelial extract-induced and SA/MeJA-induced PR gene expression, and the cis pattern of responsiveness on the osmotin promoter was the same for the mycelial extract, SA, E, or E/MeJA. Seedlings treated with P. parasitica spores in the presence of SA/MeJA were protected from pathogen colonization. However, these seedlings exhibited symptoms of cell death (disease symptoms) both in the absence and presence of P. parasitica spores, in contrast to seedlings treated with nonpathogenic mycelial extract, which remained healthy. These results suggest that the signal transduction pathways for elicitation of defense responses by exogenously applied heat-stable nonpathogenic mycelial extract and SA/MeJA overlap at the point of PR protein induction but are not identical.     

Comparison of affinity tags for protein purification

Affinity tags are highly efficient tools for purifying proteins from crude extracts. To facilitate the selection of affinity tags for purification projects, we have compared the efficiency of eight elutable affinity tags to purify proteins from Escherichia coli, yeast, Drosophila, and HeLa extracts. Our results show that the HIS, CBP, CYD (covalent yet dissociable NorpD peptide), Strep II, FLAG, HPC (heavy chain of protein C) peptide tags, and the GST and MBP protein fusion tag systems differ substantially in purity, yield, and cost. We find that the HIS tag provides good yields of tagged protein from inexpensive, high capacity resins but with only moderate purity from E. coli extracts and relatively poor purification from yeast, Drosophila, and HeLa extracts. The CBP tag produced moderate purity protein from E. coli, yeast, and Drosophila extracts, but better purity from HeLa extracts. Epitope-based tags such as FLAG and HPC produced the highest purity protein for all extracts but require expensive, low capacity resin. Our results suggest that the Strep II tag may provide an acceptable compromise of excellent purification with good yields at a moderate cost.