Effects of flunixin meglumine on pyrexia and bioenergetic variables in postparturient dairy cows.

Study objectives were to determine whether a nonsteroidal antiinflammatory drug would reduce parturition-induced inflammation and fever and consequently improve appetite, bioenergetic parameters, and production variables in transitioning dairy cows. Multiparous cows (n = 26) were randomly assigned to 1 of 2 treatments beginning at parturition: 1) flunixin meglumine (FM; 2.2 mg/kg of BW; Banamine, 50 mg/mL, Schering-Plough Animal Health, Kenilworth, NJ), or 2) saline (control) at 2.0 mL/45.5 kg of BW. All treatments were administrated i.v. daily for the first 3 d in milk (DIM). Individual milk yield and dry matter intake (DMI) were recorded daily for the first 35 DIM. Rectal temperature was measured daily at 0700 and 1600 h for the first 7 DIM. Milk composition was determined on 2, 7, 14, 21, 28, and 35 DIM and blood plasma was collected on 1, 2, 3, 4, 7, 14, 21, 28, and 35 DIM. Body weight and body condition score were determined on -7, 1, 7, 14, 21, 28, and 35 DIM. Flunixin meglumine treatment slightly increased rectal temperature (38.99 vs. 38.76 degrees C) during the first 7 DIM and reduced overall DMI (22.04 vs. 19.48 kg/d), but there were no treatment differences in overall milk yield (35.2 kg/d), 3.5% fat-corrected milk (37.6 kg/d), energy-corrected milk (37.7 kg/d), DMI (2.97% of BW), or overall energy balance (-2.32 Mcal/d). There were no treatment differences in milk fat (3.91%), protein (3.32%), or lactose (4.57%). Treatment had no effect on plasma glucose (66.5 mg/dL) or nonesterified fatty acids (553 microEq/L), but plasma urea nitrogen tended to be less in FM-treated cows (16.4 vs. 14.5 mg/dL). Daily FM administration to cows for the first 3 d after parturition slightly increased rectal temperatures by 0.23 degrees C, reduced feed intake, and did not improve production or energetic variables during the first 35 DIM in transition dairy cows.

Analyses of reproductive interactions that occur after heterospecific matings within the genus Caenorhabditis.

Formation of zygotes in internally fertilizing organisms requires a number of successful interactions between oocytes and sperm within a receptive female reproductive tract. These interactions are usually assumed to be species-specific. For most species, it is either not possible to inseminate females with sperm from a different species or not possible to observe the consequences of such an insemination because the female is opaque. Nematodes of the genus Caenorhabditis are optically transparent and prior work indicates copulation between individuals of two different species is possible. We have used a series of vital stains and other cytological methods to analyze sperm after cross-species mating. We present here a series of analyses of the postcopulatory, prefertilization interactions among three Caenorhabditis species and find that reproductive biology is conserved, to varying degrees, among all three species. This approach allows investigation into which in vivo interactions between sperm and both oocytes and the somatic gonad have been maintained during the reproductive isolation that accompanies speciation.

Myosin VI is required for asymmetric segregation of cellular components during C. elegans spermatogenesis.

BACKGROUND: The asymmetric division of cells and unequal allocation of cell contents is essential for correct development. This process of active segregation is poorly understood but in many instances has been shown to depend on the cytoskeleton. Motor proteins moving along actin filaments and microtubules are logical candidates to provide the motive force for asymmetric sorting of cell contents. The role of myosins in such processes has been suggested, but few examples of their involvement are known. RESULTS: Analysis of a Caenorhabditis elegans class VI myosin deletion mutant reveals a role for this motor protein in the segregation of cell components during spermatogenesis. Mutant spermatocytes cannot efficiently deliver mitochondria and endoplasmic reticulum/Golgi-derived fibrous-body membranous organelle complexes to budding spermatids, and fail to remove actin filaments and microtubules from the spermatids. The segregation defects are not due to a global sorting failure as nuclear inheritance is unaffected. CONCLUSIONS: C. elegans myosin VI has an important role in the unequal partitioning of both organelles and cytoskeletal components, a novel role for this class of motor protein.

Double-stranded RNA interference in Trypanosoma brucei using head-to-head promoters.

The discovery of double-stranded RNA interference (dsRNAi) in Trypanosoma brucei provides a convenient method to generate knockout phenotypes in this protozoan parasite [Ngo H, Tschudi C, Gull K, Ullu E. Double-stranded RNA induces mRNA degradation in Trypanosoma brucei. Proc Natl Acad Sci USA 1998;95:14687-14692]. The presence of double-stranded RNA (dsRNA) dominantly silences gene expression in a sequence-specific manner by causing the corresponding endogenous RNA to be degraded. To simplify the generation of knockout phenotypes in T. brucei via dsRNAi, we used two promoters arranged as an inverted repeat on a plasmid. This promoter arrangement generates transcripts of both strands of DNA inserted between the promoters, which then form dsRNA. We have used plasmids encoding either two T. brucei ribosomal RNA promoters or two bacteriophage T7 promoters to interfere with expression of alpha-tubulin (TUB), green fluorescent protein (GFP), paraflagellar rod protein A (PFRA), flagellum-adhesion glycoprotein 1 (FLA1), and histone 2B (H2B) in T. brucei. We show here that FLA1 is required for flagellar attachment in T. brucei and that H2B is required for parasite growth. Thus, the two-promoter approach efficiently generates dsRNAi in T. brucei and can be used to produce both specific and random knockout phenotypes in T. brucei. This approach should be useful in generating knockout phenotypes in other kinetoplastid parasites including Trypanosoma cruzi and Leishmania.

T lymphocyte-triggering factor of african trypanosomes is associated with the flagellar fraction of the cytoskeleton and represents a new family of proteins that are present in several divergent eukaryotes.

The trypanosome cytoskeleton consists almost entirely of microtubule-based structures. Although alpha- and beta-tubulin from Trypanosoma brucei have been well characterized, much less is known about other cytoskeleton-associated proteins in trypanosomes. Using biochemical fractionation, we demonstrate here that T lymphocyte-triggering factor (TLTF) from T. brucei is a component of the detergent-resistant and Ca(2+)-resistant fraction of the parasite cytoskeleton. This fraction contains the flagellar apparatus and a subset of cytoskeletal protein complexes that together function in cell motility, cytokinesis, and organelle inheritance. We also show that TLTF-related genes are present in several highly divergent eukaryotic organisms. Although the function of the corresponding proteins is not known, the mammalian TLTF-like gene (GAS11; growth arrest-specific gene 11) is up-regulated in growth-arrested cells and is a candidate tumor suppressor (Whitmore, S. A., Settasatian, C., Crawford, J., Lower, K. M., McCallum, B., Seshadri, R., Cornelisse, C. J., Moerland, E. W., Cleton-Jansen, A. M., Tipping, A. J., Mathew, C. G., Savnio, M., Savoia, A., Verlander, P., Auerbach, A. D., Van Berkel, C., Pronk, J. C., Doggett, N. A., and Callen, D. F. (1998) Genomics 52, 325-331), suggestive of a role in coordinating cytoskeleton activities. Consistent with this possibility, we show that the human GAS11 protein contains a 144-amino acid domain that co-localizes with microtubules when fused to the green fluorescent protein and expressed in mammalian cells. These findings suggest that TLTF represents a newly defined protein family, whose members contribute to cytoskeleton function in species as diverse as protozoa and mammals.

dpy-18 encodes an alpha-subunit of prolyl-4-hydroxylase in caenorhabditis elegans.

Collagen is an extracellular matrix (ECM) component encoded by a large multigene family in multicellular animals. Procollagen is post-translationally modified by prolyl-4-hydroxylase (EC 1.14.11.2) before secretion and participation in ECM formation. Therefore, collagen processing and regulation can be studied by examining this required interaction of prolyl-4-hydroxylase with procollagen. High-resolution polymorphism mapping was used to place the Caenorhabditis elegans dpy-18 gene on the physical map, and we show that it encodes a prolyl-4-hydroxylase alpha catalytic subunit. The Dpy phenotype of dpy-18(e364) amber mutants is more severe when this mutation is in trans to the noncomplementing deficiency tDf7, while the dpy-18(e499) deletion mutant exhibits the same phenotype as dpy-18(e499)/tDf7. Furthermore, dpy-18 RNA interference (RNAi) in wild-type worms results in Dpy progeny, while dpy-18 (RNAi) in dpy-18(e499) mutants does not alter the Dpy phenotype of their progeny. These observations suggest that the dpy-18 null phenotype is Dpy. A dpy-18::gfp promoter fusion construct is expressed throughout the hypodermis within the cells that abundantly produce the cuticle collagens, as well as in certain head and posterior neurons. While prolyl-4-hydroxylase has been studied extensively by biochemical techniques, this is the first report of a mutationally defined prolyl-4-hydroxylase in any animal.

A novel protein targeting domain directs proteins to the anterior cytoplasmic face of the flagellar pocket in African trypanosomes.

The flagellar pocket of African trypanosomes is a critical sorting station for protein and membrane trafficking, and is considered to be an Achilles' heel of this deadly pathogen. Although several proteins, including receptors for host-derived growth factors, are targeted specifically to the flagellar pocket, the signals responsible for this restricted subcellular localization are entirely unknown. Using T lymphocyte triggering factor-green fluorescent protein (TLTF(1)-GFP) fusion proteins, we demonstrate that an internal 144 amino acid domain of TLTF from Trypanosoma brucei is sufficient for directing GFP to the cytoplasmic side of the anterior flagellar pocket. Immuno-gold electron microscopy reveals that the TLTF-GFP fusion protein is located in an electron dense structure that immediately abuts the anterior flagellar pocket membrane. The amino acid sequence of the TLTF targeting domain does not resemble previously characterized protein trafficking signals, and random mutagenesis reveals that flagellar pocket targeting is conferred by a structural motif, rather than a short, contiguous array of amino acids. The aberrant sorting of two mutant proteins into the flagellum, and the targeting of a related human protein to the plus end of the trypanosome's cytoskeletal microtubules, lead us to suggest that flagellar pocket targeting involves interactions with the trypanosome cytoskeleton. The finding that TLTF-GFP is restricted to the anterior, cytoplasmic face of the flagellar pocket membrane, suggests that there is structural heterogeneity in the membrane of this organelle.

Sperm competition in the absence of fertilization in Caenorhabditis elegans.

Hermaphrodite self-fertilization is the primary mode of reproduction in the nematode Caenorhabditis elegans. However, when a hermaphrodite is crossed with a male, nearly all of the oocytes are fertilized by male-derived sperm. This sperm precedence during reproduction is due to the competitive superiority of male-derived sperm and results in a functional suppression of hermaphrodite self-fertility. In this study, mutant males that inseminate fertilization-defective sperm were used to reveal that sperm competition within a hermaphrodite does not require successful fertilization. However, sperm competition does require normal sperm motility. Additionally, sperm competition is not an absolute process because oocytes not fertilized by male-derived sperm can sometimes be fertilized by hermaphrodite-derived sperm. These results indicate that outcrossed progeny result from a wild-type cross because male-derived sperm are competitively superior and hermaphrodite-derived sperm become unavailable to oocytes. The sperm competition assays described in this study will be useful in further classifying the large number of currently identified mutations that alter sperm function and development in C. elegans.

Multiple mechanisms of immune evasion by African trypanosomes.

During infection of a mammalian host, African trypanosomes are in constant contact with the host's immune system. These protozoan parasites are infamous for their ability to evade the immune responses by periodically switching their major variant surface glycoprotein (VSG), a phenomenon called antigenic variation. Antigenic variation, however, is likely to be only one of several mechanisms enabling these organisms to thrive in the face of the immune defenses. The ability to grow in high levels of interferon-gamma (IFN-gamma) and to avoid complement-mediated destruction may also facilitate the parasite's survival. In this review we summarize (i) the activation of trypanosome genes for three different VSGs during antigenic variation, (ii) the secretion of a trypanosome protein that induces host CD8 T cells to produce IFN-gamma, and (iii) the evidence for trypanosome protein similar to a surface protease of Leishmania that plays a role in resistance to complement-mediated lysis.

The gene for a T lymphocyte triggering factor from African trypanosomes.

An early and essential event in the protective immune response against most viruses and protozoa is the production of interferon-gamma (IFN-gamma). In contrast, during infection with African trypanosomes, protozoan parasites that cause human sleeping sickness, the increased levels of IFN-gamma do not correlate with a protective response. We showed previously that African trypanosomes express a protein called T lymphocyte triggering factor (TLTF), which triggers CD8(+) T lymphocytes to proliferate and to secrete IFN-gamma. Here, we isolate the gene for TLTF and demonstrate that the recombinant version of TLTF specifically induces CD8(+), but not CD4(+), T cells to secrete IFN-gamma. Studies with TLTF fused to the green fluorescent protein show that TLTF is localized to small vesicles that are found primarily at or near the flagellar pocket, the site of secretion in trypanosomes. TLTF is likely to be only the first example of a class of proteins that we designate as trypanokines, i.e., factors secreted by trypanosomes that modulate the cytokine network of the host immune system for the benefit of the parasite.

Actin and myosin function in directed vacuole movement during cell division in Saccharomyces cerevisiae.

During cell division, cytoplasmic organelles are not synthesized de novo, rather they are replicated and partitioned between daughter cells. Partitioning of the vacuole in the budding yeast Saccharomyces cerevisiae is coordinated with the cell cycle and involves a dramatic translocation of a portion of the parental organelle from the mother cell into the bud. While the molecular mechanisms that mediate this event are unknown, the vacuole's rapid and directed movements suggest cytoskeleton involvement. To identify cytoskeletal components that function in this process, vacuole inheritance was examined in a collection of actin mutants. Six strains were identified as being defective in vacuole inheritance. Tetrad analysis verified that the defect cosegregates with the mutant actin gene. One strain with a deletion in a myosin-binding region was analyzed further. The vacuole inheritance defect in this strain appears to result from the loss of a specific actin function; the actin cytoskeleton is intact and protein targeting to the vacuole is normal. Consistent with these findings, a mutation in the actin-binding domain of Myo2p, a class V unconventional myosin, abolishes vacuole inheritance. This suggests that Myo2p serves as a molecular motor for vacuole transport along actin filaments. The location of actin and Myo2p relative to the vacuole membrane is consistent with this model. Additional studies suggest that the actin filaments used for vacuole transport are dynamic, and that profilin plays a critical role in regulating their assembly. These results present the first demonstration that specific cytoskeletal proteins function in vacuole inheritance.

Regulated copper uptake in Chlamydomonas reinhardtii in response to copper availability.

A saturable and temperature-dependent copper uptake pathway has been identified in Chlamydomonas reinhardtii. The uptake system has a high affinity for copper ions (Km approximately 0.2 microM) and is more active in cells that are adapted to copper deficiency than to cells grown in a medium containing physiological (submicromolar to micromolar) copper ion concentrations. The maximum velocity of copper uptake by copper-deficient cells (169 pmol h-1 10(6) cells-1 or 62 ng min-1 mg-1 chlorophyll) is up to 20-fold greater than that of fully copper-supplemented cells, and the Km (approximately 2 x 10(2) nM) is unaffected. Thus, the same uptake system appears to operate in both copper-replete and copper-deficient cells, but its expression or activity must be induced under copper-deficient conditions. A cupric reductase activity is also increased in copper-deficient compared with copper-sufficient cells. The physiological characteristics of the regulation of this cupric reductase are compatible with its involvement in the uptake pathway. Despite the operation of the uptake pathway under both copper-replete and copper-deficient conditions, C. reinhardtii cells maintained in fully copper-supplemented cells do not accumulate copper in excess of their metabolic need. These results provide evidence for a homeostatic mechanism for copper metabolism in C. reinhardtii.

Coordinate expression of coproporphyrinogen oxidase and cytochrome c6 in the green alga Chlamydomonas reinhardtii in response to changes in copper availability.

To maintain photosynthetic competence under copper-deficient conditions, the green alga Chlamydomonas reinhardtii substitutes a heme protein (cytochrome c6) for an otherwise essential copper protein, viz. plastocyanin. Here, we report that the gene encoding coproporphyrinogen oxidase, an enzyme in the heme biosynthetic pathway, is coordinately expressed with cytochrome c6 in response to changes in copper availability. We have purified coproporphyrinogen oxidase from copper-deficient C.reinhardtii cells, and have cloned a cDNA fragment which encodes it. Northern hybridization analysis confirmed that the protein is nuclear-encoded and that, like cytochrome c6, its expression is regulated by copper at the level of mRNA accumulation. The copper-responsive expression of coproporphyrinogen oxidase parallels cytochrome c6 expression exactly. Specifically, the copper-sensing range and metal selectivity of the regulatory components, as well as the time course of the responses, are identical. Hence, we propose that the expression of these two proteins is controlled by the same metalloregulatory mechanism. Our findings represent a novel metalloregulatory response in which the synthesis of one redox cofactor (heme) is controlled by the availability of another (Cu).

Infrainguinal directional atherectomy: long-term follow-up and comparison with percutaneous transluminal angioplasty.

PURPOSE: To assess the long-term results of directional atherectomy (DA) for femoropopliteal artery atherosclerotic lesions and to compare the results to those previously reported for percutaneous transluminal balloon angioplasty (PTA). MATERIALS AND METHODS: Eight-four percutaneous DA procedures performed on 75 patients between July 1988 and August 1992 were retrospectively reviewed and evaluated for technical and initial clinical success. Long-term patency was assessed with a combination of ankle-brachial index measurements and angiography. RESULTS: Initial technical success was achieved in 77 of 84 procedures (92%). Follow-up of 72 patients was obtained, including 74 of the 84 (88%) DA procedures with a mean follow-up of 17.4 months (range 1-48 months). Primary patency was 78% at 1 year and 57% at 2 years. Patients with diabetes, complete luminal occlusion, or limb salvage situations had significantly lower patency. CONCLUSIONS: Femoropopliteal artery DA can be performed safely with a high technical and initial clinical success. Long-term patency is improved when compared with published series for PTA. With this improvement in mind, DA may have a place in the treatment of focal infrainguinal stenoses.

In Vivo Competition between Plastocyanin and a Copper-Dependent Regulator of the Chlamydomonas reinhardtii Cytochrome c(6) Gene.

The Chlamydomonas reinhardtii gene encoding cytochrome c(6) (Cyt c(6)) is transcriptionally repressed by cupric ions. In quantitating the level of expression of this gene as a function of cupric ions available per cell, we find that transformed Chlamydomonas reinhardtii cells that accumulate high levels of plastocyanin (a type I copper protein) have a higher sensory threshold for copper-dependent repression of the Cyt c(6) gene than do untransformed, otherwise isogenic, cells that are plastocyanin-deficient. Also, in wild-type cells, the extent to which the gene is expressed at any given ratio of copper/cell is exactly correlated with the predicted deficiency (at this level of copper) in the organism's capacity to synthesize holoplastocyanin. These results support a simple model in which the sensory threshold for transcriptional repression of the Cyt c(6) gene is determined by direct competition for intracellular copper ions between a copper-binding regulator of this gene and plastocyanin. Thus, the organism is able to maintain a constant amount of Cyt c(6) plus plastocyanin per Photosystem I. With the use of in vitro-generated Cyt c(6)-encoding transcripts as a standard for the quantitation of cellular Cyt c(6) mRNA levels, we estimate that whereas copper-deficient wild-type cells maintain approximately 1 x 10(2) to 4 x 10(2) Cyt c(6)-specific transcripts per cell, copper-supplemented cells contain, on average, less than one Cyt c(6)-encoding mRNA. Thus, repression of the Cyt c(6) gene by copper ions is essentially 100%, making it unlikely that Cyt c(6) has any essential metabolic function in copper-supplemented cells. We find also that the steady-state levels of several transcripts, including those for Cyt c(6), are influenced by cell density, so that cells harvested at low density contain several-fold as many copies of a particular message as cells harvested near stationary phase.

Contemporary medical management of left ventricular dysfunction and congestive heart failure.

OBJECTIVE: The primary purpose of this review was to address the following question: based on the best available evidence, what should be the current medical management of congestive heart failure (CHF)? DATA SOURCES: The major sources for this review were from searches of the English language literature, including computer and bibliography reviews, of all randomized, controlled clinical trials and overview analyses of positive inotropic agents, preload/afterload reduction agents and beta-blocker medications in CHF. STUDY SELECTION: The number of studies reviewed was approximately 40. The major criterion for selection was that the studies be of CHF patients in randomized controlled clinical trials, particularly with a mortality/survival endpoint. Additional clinical trials of nonmortality endpoints in CHF patients and mortality trials in non-CHF patients were also selected to support possible pathophysiological insights for future CHF trials. DATA EXTRACTION: The data, particularly for the accompanying tables, were initially extracted by a single reviewer using common qualitative guidelines as far as was possible within the different temporal, etiological and geographic frameworks of the original component studies. Conclusions are drawn from this data synthesis and from published overviews. DATA SYNTHESIS: Angiotensin converting enzyme (ACE) inhibition therapy is effective in reducing mortality and morbidity in severe left ventricular dysfunction and CHF. Other systemic vasodilators may also be beneficial. The effects of digitalis on survival and morbidity in CHF are presently uncertain, but should be resolved in the near future. Other inotropic agents, at least in the long term, are clinically detrimental. Diuretics decrease morbidity, but their effect on mortality in CHF remains unknown. Beta-blocker and magnesium therapy offer promise in CHF, but await definitive clinical trials evaluation. CONCLUSIONS: The current medical therapy of CHF should definitely include ACE inhibitors, probably diuretics and possibly other vasodilators. Further viable trials of promising new, and older heretofore under-evaluated, CHF therapies are needed. Additionally, innovative strategies are needed to deal with this disease which has an increasing prevalence. Two strategies, primary prevention of CHF and a 'Heart Function Clinic', are discussed.

Isolation and structural characterization of the Chlamydomonas reinhardtii gene for cytochrome c6. Analysis of the kinetics and metal specificity of its copper-responsive expression.

We have isolated a 5-kilobase pair fragment of genomic DNA containing the entire coding region for the Chlamydomonas reinhardtii gene encoding the copper-repressible Cyt c6. A region comprising 2.6 kilobase pairs contains the entire transcribed region plus 852 nucleotides upstream of the Cyt c6 transcription start site and 495 nucleotides downstream of the conserved C. reinhardtii polyadenylation signal. Comparison of the genomic sequence with the cDNA sequence (Merchant, S., and Bogorad, L. (1987) J. Biol. Chem. 262, 9062-9067) revealed that the coding region is interrupted by two introns, each of which is flanked by C. reinhardtii consensus intron/exon boundaries. Primer extension and S1 nuclease protection analyses identified the 5' border of the Cyt c6 mRNA at approximately 79 base pairs upstream from the initiator methionine. Analysis of the 5' upstream region reveals no significant similarity to sequences found in upstream regions of other copper-regulated genes. Time-course studies indicate that 1) the mature Cyt c6 mRNA has a half-life of approximately 45-60 min and is completely lost within 4 h, and 2) the primary, unspliced transcript has a half-life of approximately 10 min and is completely lost within 30 min after the addition of copper ions to copper-depleted cells. These results indicate that the response to copper occurs very rapidly upon elevation of extracellular copper levels. Although this gene is unresponsive to silver ions in vivo, in contrast to the yeast copper-responsive CUP1 gene (Furst, P., Hu, S., Hackett, R., and Hamer, D. (1988) Cell 55, 705-717), it does respond to mercury ions, albeit with less sensitivity. Mercury ions cannot, however, substitute for copper in allowing the accumulation of plastocyanin in vivo.

Phosphotransferase system of Staphylococcus aureus: its requirement for the accumulation and metabolism of galactosides.

The phosphotransferase system of Staphylococcus aureus was characterized. Mutants defective in enzyme I and heat-stable (HPr) protein as well as in the two components specific to lactose accumulation, factor III and enzyme II, were isolated. Colorimetric assays for each of the components are presented based on the formation of o-nitrophenyl-beta-d-galactoside-6-phosphate by the system and its hydrolysis by the staphylococcal 6-phospho-beta-galactosidase. The components were partially purified and their molecular weights were estimated: enzyme I, 100,000 +/- 15%; HPr, 10,000 +/- 15%; factor III, 30,000 +/- 15%; 6-phospho-beta-galactosidase, 45,000. Enzyme II is a membrane-bound protein.