Antimicrobial use and stewardship practices on Australian beef feedlots.

OBJECTIVE: Improving antimicrobial stewardship in the livestock sector requires an understanding of the motivations for antimicrobial use and the quantities consumed. However,detailed information on antimicrobial use in livestock sectors is lacking. This cross-sectional study aimed to better understand antimicrobial use in the beef feedlot sector in Australia. DESIGN: A self-administered questionnaire asking about antimicrobial use and reasons for use was designed and mailed to beef feedlot operators in Australia. Respondents were asked to report the percentage of animals treated, purpose of use, and disease conditions targeted for 26antimicrobial agents. RESULTS: In total, 83 of 517 (16.1%) beef feedlot operators completed the survey. Monensin (61.0%of respondents) and virginiamycin (19.5%of respondents) were the most commonly reported in-feed antimicrobials. In-feed antimicrobial agents were most frequently used by respondents for treatment of gastrointestinal diseases (52.8%). Antimicrobials were used for growth promotion by 42.1% of respondents, with most (85.7%) reporting the use of ionophores(a group of compounds not used in human medicine). Short-acting penicillin(69.1%), short-acting oxytetracycline, and tulathromycin (both 57.3%) werethe most common injectable antimicrobial agents used. Injectable antimicrobials were most frequently used to treat respiratory (72.3%) and musculoskeletal (67.5%) conditions. CONCLUSION: Overall,the use of antimicrobials was appropriate for the purpose indicated, and there was a strong preference for drugs of low-importance in human medicine. The data described here stand to be a strong influence on the implementation of an antimicrobial stewardship program in the sector.

Esperanto for histones: CENP-A, not CenH3, is the centromeric histone H3 variant.

The first centromeric protein identified in any species was CENP-A, a divergent member of the histone H3 family that was recognised by autoantibodies from patients with scleroderma-spectrum disease. It has recently been suggested to rename this protein CenH3. Here, we argue that the original name should be maintained both because it is the basis of a long established nomenclature for centromere proteins and because it avoids confusion due to the presence of canonical histone H3 at centromeres.

First Report of White Pine Blister Rust on Rocky Mountain Bristlecone Pine.

White pine blister rust caused by Cronartium ribicola was introduced into North America in the early 20th century and is spreading throughout the range of five-needle pines. In northern Colorado, this pathogen was first observed in 1998 on limber pine (Pinus flexilis) (1). It has not been reported on Rocky Mountain or Great Basin bristlecone pine (Pinus aristata and P. longaeva, respectively) in nature. However, Rocky Mountain bristlecone pine is susceptible to the disease when artificially inoculated (2). In October 2003, a Rocky Mountain bristlecone pine was found infected with C. ribicola in the Great Sand Dunes National Monument, Alamosa County, Colorado. Seven branch cankers were observed on the tree. Cankers ranged in length from 15 to 41 cm and were estimated to be approximately 5 to 7 years old. Distinct C. ribicola branch symptoms were observed, including flagging, spindle-shaped swellings, and 6 mm long aecial scars. A branch was deposited at the Colorado State Herbarium. Microscopic examination of spores within remnant aecial blisters revealed aeciospores characteristic of C. ribicola (yellow-orange, ellipsoid, verrucose, and 19 × 25 µm). Cankers were only observed on one bristlecone pine. However, most limber pines in the area were infected with C. ribicola, including a limber pine less than 1 m from the infected bristlecone pine. To our knowledge, this is the first report that shows Rocky Mountain bristlecone pine can become infected naturally, and the pathogen is further south in Colorado on limber pine than previously reported. These observations suggest the need for a more complete investigation of this disease on bristlecone pines. References: (1) D. W. Johnson and W. R. Jacobi. Plant Dis. 84:595, 2000. (2) B. R. Stephan, Allg. Forst Z. 28:695, 1985.

Specification of kinetochore-forming chromatin by the histone H3 variant CENP-A.

The mechanisms that specify precisely where mammalian kinetochores form within arrays of centromeric heterochromatin remain largely unknown. Localization of CENP-A exclusively beneath kinetochore plates suggests that this distinctive histone might direct kinetochore formation by altering the structure of heterochromatin within a sub-region of the centromere. To test this hypothesis, we experimentally mistargeted CENP-A to non-centromeric regions of chromatin and determined whether other centromere-kinetochore components were recruited. CENP-A-containing non-centromeric chromatin assembles a subset of centromere-kinetochore components, including CENP-C, hSMC1, and HZwint-1 by a mechanism that requires the unique CENP-A N-terminal tail. The sequence-specific DNA-binding protein CENP-B and the microtubule-associated proteins CENP-E and HZW10 were not recruited, and neocentromeric activity was not detected. Experimental mistargeting of CENP-A to inactive centromeres or to acentric double-minute chromosomes was also not sufficient to assemble complete kinetochore activity. The recruitment of centromere-kinetochore proteins to chromatin appears to be a unique function of CENP-A, as the mistargeting of other components was not sufficient for assembly of the same complex. Our results indicate at least two distinct steps in kinetochore assembly: (1) precise targeting of CENP-A, which is sufficient to assemble components of a centromere-prekinetochore scaffold; and (2) targeting of kinetochore microtubule-associated proteins by an additional mechanism present only at active centromeres.

Animal cytokinesis: breaking up is hard to do.

Recent studies have shed new light on how the physical association between sister cells is severed at the end of cytokinesis while the membrane is resealed. Comparisons with yeast suggest that daughter cell shape may feed back to regulate cytokinesis through the Bub2 checkpoint system.

Large-scale chromatin fibers of living cells display a discontinuous functional organization.

We investigated the chromatin organization of living cells with a combination of recently developed approaches for histone and DNA labeling. Nucleosomal DNA was labeled with a histone H2B-GFP (green fluorescent protein) fusion protein and the chromatin organization of living HeLa cells was analyzed by high resolution confocal microscopy. Within the perinuclear and perinucleolar regions chromatin was organized into large-scale fibers of 2 to 8 microm in length and 300 to 500 nm in diameter. Within the nuclear interior we observed similar large-scale fibers, but in addition focal as well as diffuse forms of organization. Comparison with standard labeling and detection procedures revealed major differences in the chromatin organization observed. Chromatin organization revealed by the distribution of histone H2B-GFP was directly compared with the functional organization of chromatin by Cy3-dUTP labeling of DNA replicating at a specific time. DNA regions replicating at a specific time display characteristic physical and functional properties. Analysis of Cy3-labeled foci revealed that they are associated with all three forms of chromatin organization (fibrillar, focal and diffuse). In particular, Cy3-labeled foci appeared as discontinuous regions of large-scale fibers. These results demonstrate that large-scale chromatin fibers have discontinuous functional characteristics.

A solid foundation: functional specialization of centromeric chromatin.

Centromeres provide a distinctive mechanical function for the chromosomes as the site of kinetochore assembly and force generation in mitosis and meiosis. Recent studies show that a unique form of chromatin, based on the histone-H3-like protein CENP-A and homologues, provides a conserved foundation for this mechanical chromatin domain. CENP-A plays a role in templating kinetochore assembly and may be a central element in the epigenetic maintenance of centromere identity. Cohesion at the centromere, intimately linked to kinetochore assembly, is required for integrating spindle forces exerted across the centromere and for establishing the bipolar geometry of sister kinetochores.

Differential regulation of CENP-A and histone H3 phosphorylation in G2/M.

After DNA replication, cells condense their chromosomes in order to segregate them during mitosis. The condensation process as well as subsequent segregation requires phosphorylation of histone H3 at serine 10. Histone H3 phosphorylation initiates during G2 in pericentric foci prior to H3 phosphorylation in the chromosome arms. Centromere protein A (CENP-A), a histone H3-like protein found uniquely at centromeres, contains a sequence motif similar to that around H3 Ser10, suggesting that CENP-A phosphorylation might be linked to pericentric initiation of histone H3 phosphorylation. To test this hypothesis, we generated peptide antibodies against the putative phosphorylation site of CENP-A. ELISA, western blot and immunocytochemical analyses show that CENP-A is phosphorylated at the shared motif. Simultaneous co-detection demonstrates that phosphorylation of CENP-A and histone H3 are separate events in G2/M. CENP-A phosphorylation occurs after both pericentric initiation and genome-wide stages of histone H3 phosphorylation. Quantitative immunocytochemistry reveals that CENP-A phosphorylation begins in prophase and reaches maximal levels in prometaphase. CENP-A phosphoepitope reactivity is lost during anaphase and becomes undetectable in telophase cells. Duplication of prekinetochores, detected as the doubling of CENP-A foci, occurs prior to complete histone H3 phosphorylation in G2. Mitotic phosphorylation of histone H3-family proteins shows tight spatial and temporal control, occurring in three phases: (1) pericentric H3 phosphorylation, (2) chromosome arm H3 phosphorylation and (3) CENP-A phosphorylation at kinetochores. These observations reveal new cytological landmarks characteristic of G2 progression.

Degradation of nucleosome-associated centromeric histone H3-like protein CENP-A induced by herpes simplex virus type 1 protein ICP0.

Cells infected by herpes simplex virus type 1 in the G2 phase of the cell cycle become stalled at an unusual stage of mitosis defined as pseudoprometaphase. This block correlates with the viral immediate-early protein ICP0-induced degradation of the centromere protein CENP-C. However, the observed pseudoprometaphase phenotype of infected mitotic cells suggests that the stability of other centromere proteins may also be affected. Here, we demonstrate that ICP0 also induces the proteasome-dependent degradation of the centromere protein CENP-A. By a series of Western blot and immunofluorescence experiments we show that the endogenous 17-kDa CENP-A and an exogenous tagged version of CENP-A are lost from centromeres and degraded in infected and transfected cells as a result of ICP0 expression. CENP-A is a histone H3-like protein associated with nucleosome structures in the inner plate of the kinetochore. Unlike fully transcribed lytic viral DNA, the transcriptionally repressed latent herpes simplex virus type 1 genome has been reported to have a nucleosomal structure similar to that of cellular chromatin. Because ICP0 plays an essential part in controlling the balance between the lytic and latent outcomes of infection, the ICP0-induced degradation of CENP-A is an intriguing feature connecting different aspects of viral and/or cellular genome regulation.

CENP-A is phosphorylated by Aurora B kinase and plays an unexpected role in completion of cytokinesis.

Aurora B is a mitotic protein kinase that phosphorylates histone H3, behaves as a chromosomal passenger protein, and functions in cytokinesis. We investigated a role for Aurora B with respect to human centromere protein A (CENP-A), a centromeric histone H3 homologue. Aurora B concentrates at centromeres in early G2, associates with histone H3 and centromeres at the times when histone H3 and CENP-A are phosphorylated, and phosphorylates histone H3 and CENP-A in vitro at a similar target serine residue. Dominant negative phosphorylation site mutants of CENP-A result in a delay at the terminal stage of cytokinesis (cell separation). The only molecular defects detected in analysis of 22 chromosomal, spindle, and regulatory proteins were disruptions in localization of inner centromere protein (INCENP), Aurora B, and a putative partner phosphatase, PP1gamma1. Our data support a model where CENP-A phosphorylation is involved in regulating Aurora B, INCENP, and PP1gamma1 targeting within the cell. These experiments identify an unexpected role for the kinetochore in regulation of cytokinesis.

Chromatin assembly at kinetochores is uncoupled from DNA replication.

The specification of metazoan centromeres does not depend strictly on centromeric DNA sequences, but also requires epigenetic factors. The mechanistic basis for establishing a centromeric "state" on the DNA remains unclear. In this work, we have directly examined replication timing of the prekinetochore domain of human chromosomes. Kinetochores were labeled by expression of epitope-tagged CENP-A, which stably marks prekinetochore domains in human cells. By immunoprecipitating CENP-A mononucleosomes from synchronized cells pulsed with [(3)H]thymidine we demonstrate that CENP-A-associated DNA is replicated in mid-to-late S phase. Cytological analysis of DNA replication further demonstrated that centromeres replicate asynchronously in parallel with numerous other genomic regions. In contrast, quantitative Western blot analysis demonstrates that CENP-A protein synthesis occurs later, in G2. Quantitative fluorescence microscopy and transient transfection in the presence of aphidicolin, an inhibitor of DNA replication, show that CENP-A can assemble into centromeres in the absence of DNA replication. Thus, unlike most genomic chromatin, histone synthesis and assembly are uncoupled from DNA replication at the kinetochore. Uncoupling DNA replication from CENP-A synthesis suggests that regulated chromatin assembly or remodeling could play a role in epigenetic centromere propagation.

Annexation of the interchromosomal space during viral infection.

The nucleus is known to be compartmentalized into units of function, but the processes leading to the spatial organization of chromosomes and nuclear compartments are not yet well defined. Here we report direct quantitative analysis of the global structural perturbations of interphase chromosome and interchromosome domain distribution caused by infection with herpes simplex virus-1 (HSV-1). Our results show that the peripheral displacement of host chromosomes that correlates with expansion of the viral replication compartment (VRC) is coupled to a twofold increase in nuclear volume. Live cell dynamic measurements suggest that viral compartment formation is driven by the functional activity of viral components and underscore the significance of spatial regulation of nuclear activities.

CENP-E forms a link between attachment of spindle microtubules to kinetochores and the mitotic checkpoint.

Here we show that suppression of synthesis of the microtubule motor CENP-E (centromere-associated protein E), a component of the kinetochore corona fibres of mammalian centromeres, yields chromosomes that are chronically mono-orientated, with spindles that are flattened along the plane of the substrate. Despite apparently normal microtubule numbers and the continued presence at kinetochores of other microtubule motors, spindle poles fragment in the absence of CENP-E, which implicates this protein in delivery of components from kinetochores to poles. CENP-E represents a link between attachment of spindle microtubules and the mitotic checkpoint signalling cascade, as depletion of this motor leads to profound checkpoint activation, whereas immunoprecipitation reveals a nearly stoichiometric association of CENP-E with the checkpoint kinase BubR1 during mitosis.

First Report of White Pine Blister Rust in Nevada.

White pine blister rust, caused by Cronartium ribicola Fisch., was found in 1997 infecting white pines (genus Pinus, subgenus Strobus) at two locations in the Carson Range of western Nevada. Rust incidence, infection age, damage to trees, rust phenology, and host distribution were evaluated at one of these locations and a nearby location in California in 1998. At the first location (39.3°N, 119.9°W), C. ribicola was found infecting 24 of 49 whitebark pines (P. albicaulis Engelm.) near Mt. Rose Summit on Highway 27 at 2,710 m elevation, ≈6 km northeast of Incline Village, Washoe County, NV. Among infected trees, 33% had only branch cankers, 54% had live stem cankers, and 12% had stem cankers that had killed portions of trees distal to cankers. No trees had died from infection. At the second location (39.1°N, 119.9°W), we found only 6 of 50 (12%) infected western white pines (P. monticola Dougl.) near Genoa Peak (≈2,750 m elevation), 3 km east of Lake Tahoe, Douglas County, NV; however, stem cankers occurred on 4 of the 6 infected trees. In September 1998, whitebark pines at Mt. Rose and Tahoe Meadows (2,550 m elevation, 1.5 km southwest of Mt. Rose Summit) were examined, and the following was observed: (i) aeciospore production was at its peak, indicating that sporulation can occur exceptionally late in the season in this region; (ii) signs of blister rust infection were absent on the telial hosts of C. ribicola (Ribes cereum, R. montigenum, and R. nevadense) at both locations; (iii) ≈80% of the cankers occurred on host wood produced in 1978 and 1979; and (iv) the oldest cankers originated on wood produced in 1968 and the youngest on wood produced in 1980. In October 1998, infected western white pines were examined at a location (2,650 m elevation) ≈30 km north of Lake Tahoe on Babbitt Peak, Sierra County, CA (39.6°N, 120.1°W). At this location, no trees had died from infection, fresh aeciospores were abundant on live cankers, R. montigenum and R. cereum were present but did not show signs of infection, and 19 of 20 cankers examined were on wood produced between 1978 and 1980. White pine blister rust was not found at any of 10 other locations examined throughout Nevada from 1995 to 1997. This is believed to be the first documented report of C. ribicola infecting white pines in Nevada and the easternmost extension of blister rust in the Sierra Nevada Region. These observations suggest that our understanding of blister rust spread and infection dynamics east of the Sierra Nevada crest is incomplete and that future surveys and research in this region must address, among other issues, timing of aeciospore production on pine, and the possibility of blister rust spread into the Great Basin.

CENP-A associated complex satellite DNA in the kinetochore of the Indian muntjac.

The centromere/kinetochore complex is a chromosomal assembly that mediates chromosome motility and mitotic regulation by interacting with microtubules of the mitotic spindle apparatus. Centromere protein A (CENP-A) is a histone H3 homolog that is concentrated in the chromatin of the inner kinetochore plate of human chromosomes. To identify DNA sequences associated with the inner kinetochore plate, we used anticentromere autoantibodies to immunoprecipitate CENP-A associated chromatin selectively from Indian muntjac fibroblasts. DNA was cloned from immunoprecipitated CENP-A- associated chromatin and characterized by DNA sequence and hybridization analyses. A novel centromeric satellite DNA sequence was identified and shown by fluorescence in situ hybridization analysis to be present at all centromeres of the Indian muntjac. This satellite DNA constitutes a 972 bp monomer repeat and shows partial homology with satellite II DNA of the white-tailed deer. Southern blot analysis of muntjac genomic DNA suggests that this satellite DNA is present in repetitive tandem arrays and contains complex internal arrangements. In conjunction with previous work showing the association of CENP-A with human alpha-satellite DNA, we conclude that the mammalian inner kinetochore plate contains a unique form of chromatin that contains CENP-A in association with complex satellite DNA.

Using time-lapse confocal microscopy for analysis of centromere dynamics in human cells.

Using these methods we have shown that the alpha-satellite domain of the human centromere behaves as an elastic element, stretching in response to spindle forces applied during prometaphase and metaphase (Shelby et al., 1996). These data complement previous observations of centromere stretching during mitosis (e.g., Skibbens et al., 1993) by demonstrating a specific molecular compartment within the centromere, the satellite heterochromatin domain, that supports this strain. Centromere stretching reports on the net force applied across the centromere during mitosis and the availability of a fluorescence-based assay system in human cells provides a robust assay system to complement the elegant DIC-based methods that have been perfected using marsupial and newt cell cultures (Cassimeris et al., 1990; Skibbens et al., 1993, 1995; Rieder et al., 1994). Current applications of this method are directed toward examining the relationship between centromere tension and microtubule dynamics using pharmacological approaches and the behavior of kinetochore-associated regulatory proteins, such as Mad2 and Bub1 (Li and Benezra, 1996; Chen et al., 1996; Taylor and McKeon, 1997), as a function of centromere distortion. In addition, GFP-labeled centromeres can be observed during interphase, providing a novel window into chromosome organization within the nucleus. Our observations show that centromeres distribute into the newly forming nucleus at telophase by what is apparently a uniform isometric expansion, with little evidence for directed motion of individual centromeres contributing to the formation of the G1 nucleus. During interphase, centromeres show very little movement in general, behaving as though embedded in a rigid matrix. Sustained movements of individual centromeres or groups of centromeres are occasionally observed, however, suggesting that chromosome position is subject to change during interphase (Shelby et al., 1996). These experiments complement those described by Belmont and colleagues, who have developed a method to mark specific chromosomal sites with GFP for analysis in vivo (see Chapter 13; Robinett et al., 1996; Straight et al., 1996). These new GFP-based techniques for direct observation of defined DNA sequence domains in vivo carry the logic of in situ hybridization analysis into living cells and, coupled with new methods for observing global chromatin architecture as well as functional nuclear protein domains (Huang et al., 1997; Misteli et al., 1997), promise significant progress toward understanding the dynamic organization of the genome within the living nucleus.

The mammalian centromere: structural domains and the attenuation of chromatin modeling.

The centromere-kinetochore complex can be divided into distinct domains based on structure and function. Previous work has used CREST auto-antibodies with various microscopic techniques to map the locations of proteins within the centromere-kinetochore complex and to analyze the maturation of prekinetochores before mitosis. Here we have focused on the centromere-specific histone Centromere Protein (CENP)-A and its spatial relationship to other histones and histone modifications found in condensed chromatin. We demonstrate that the phosphorylation of histone H3 is essentially excluded from a specific region of centromeric chromatin, defined by the presence of CENP-A. Interspersion of CENP-B with phosphorylated H3 in the inner centromere indicates that the exclusion of H3 modification is not a general property of alpha-satellite DNA. We also demonstrate that these regions are functionally distinct by fragmenting mitotic chromatin into motile centromere-kinetochore fragments that contain CENP-A with little or no phosphorylated H3 and nonmotile fragments that contain exclusively phosphorylated H3. The sequence of CENP-A diverges from H3 in a number of key residues involved in chromosome condensation and in transcription, potentially allowing a more specialized chromatin structure within centromeric heterochromatin, on which kinetochore plates may nucleate and mature. This specialized centromere subdomain would be predicted to have a very tight and static nucleosome structure as a result of the absence of H3 phosphorylation and acetylation.

Histone-GFP fusion protein enables sensitive analysis of chromosome dynamics in living mammalian cells.

BACKGROUND: The amplification of oncogenes in cancer cells is often mediated by paired acentric chromatin bodies called double minute chromosomes (DMs), which can accumulate to a high copy number because of their autonomous replication during the DNA synthesis phase of the cell cycle and their subsequent uneven distribution to daughter cells during mitosis. The mechanisms that control DM segregation have been difficult to investigate, however, as the direct visualization of DMs in living cells has been precluded because they are far smaller than normal chromosomes. We have visualized DMs by developing a highly sensitive method for observing chromosome dynamics in living cells. RESULTS: The human histone H2B gene was fused to the gene encoding the green fluorescent protein (GFP) of Aequorea victoria and transfected into human HeLa cells to generate a stable line constitutively expressing H2B-GFP. The H2B-GFP fusion protein was incorporated into nucleosomes without affecting cell cycle progression. Using confocal microscopy, H2B-GFP allowed high-resolution imaging of both mitotic chromosomes and interphase chromatin, and the latter revealed various chromatin condensation states in live cells. Using H2B-GFP, we could directly observe DMs in living cancer cells; DMs often clustered during anaphase, and could form chromosomal 'bridges' between segregating daughter chromosomes. Cytokinesis severed DM bridges, resulting in the uneven distribution of DMs to daughter cells. CONCLUSIONS: The H2B-GFP system allows the high-resolution imaging of chromosomes, including DMs, without compromising nuclear and chromosomal structures and has revealed the distinctive clustering behavior of DMs in mitotic cells which contributes to their asymmetric distribution to daughter cells.

Isolation and comparison of natural and recombinant human CENP-A autoantigen.

Anticentromere antibodies (ACA) are associated with systemic sclerosis (scleroderma) patients exhibiting the more benign or so called limited manifestation of the disease (lSSc). ACA reactivity is directed against multiple polypeptide targets, the smallest of which is designated CENP-A. CENP-A is not an abundant cellular constituent; therefore to maximize recovery, we developed a protocol with a minimum of steps to isolate CENP-A from a human cell line. The trace cellular amount of this protein clearly dictated the production of its recombinant counterpart to facilitate determination of the role of the CENP-A antigen in scleroderma pathogenesis. Here we describe the eukaryotic expression of CENP-A cDNA using baculovirus-mediated infection of insect cells. The non-fusion recombinant protein spans the natural residues of the human CENP-A protein and rCENP-A followed the same chromotographic sequence for purification as did the natural source. The availability of the bona fide antigen provided a critical standard upon which to document authenticity of the recombinant polypeptide. The two forms of this antigen have been compared and shown to exhibit similar physical and antigenic properties.