Development of mass allelic exchange, a technique to enable sexual genetics in Escherichia coli.

Despite dramatic advances in genomics, connecting genotypes to phenotypes is still challenging. Sexual genetics combined with linkage analysis is a powerful solution to this problem but generally unavailable in bacteria. We build upon a strong negative selection system to invent mass allelic exchange (MAE), which enables hybridization of arbitrary (including pathogenic) strains of Escherichia coli. MAE reimplements the natural phenomenon of random cross-overs, enabling classical linkage analysis. We demonstrate the utility of MAE with virulence-related gain-of-function screens, discovering that transfer of a single operon from a uropathogenic strain is sufficient for enabling a commensal E. coli to form large intracellular bacterial collections within bladder epithelial cells. MAE thus enables assaying natural allelic variation in E. coli (and potentially other bacteria), complementing existing loss-of-function genomic techniques.

Time-resolved nonlinear optical spectroscopy of perovskites.

Ultrafast decay of optical phonons has been studied in wide-bandgap BaSnO3 and SrTiO3 perovskites using nonlinear spectroscopy with 120 femtosecond time resolution. The coherent Raman mode excitations have been selected and traced with tunable optical pulses. Decay of symmetry forbidden modes of vibrations have been detected directly in time. Phonon decay rates for the main LO- and TO- phonon modes have been found to be within 1.36-1.78 ps-1 and are explained in terms of parametric phonon interactions and pure dephasing mechanisms in the materials that are of interest in microelectronic applications.

Femtosecond coherent Raman system with >75†dB dynamic range for probing vibration modes across 250-2400†cm-1.

We report on the design and performance of a time-resolved Coherent Raman spectroscopy system with time resolution of better than 120 fs. The coherent transients can be traced with more than 75 dB dynamic range while accessing and probing Raman active modes across a 250-2400 cm-1 frequency. The system delivers an equivalent spectral resolution of better than 0.1 cm-1 regarding line bandwidth parameters for probed Raman resonances.

Hypoxic pulmonary vasoconstriction.

It has been known for more than 60 years, and suspected for over 100, that alveolar hypoxia causes pulmonary vasoconstriction by means of mechanisms local to the lung. For the last 20 years, it has been clear that the essential sensor, transduction, and effector mechanisms responsible for hypoxic pulmonary vasoconstriction (HPV) reside in the pulmonary arterial smooth muscle cell. The main focus of this review is the cellular and molecular work performed to clarify these intrinsic mechanisms and to determine how they are facilitated and inhibited by the extrinsic influences of other cells. Because the interaction of intrinsic and extrinsic mechanisms is likely to shape expression of HPV in vivo, we relate results obtained in cells to HPV in more intact preparations, such as intact and isolated lungs and isolated pulmonary vessels. Finally, we evaluate evidence regarding the contribution of HPV to the physiological and pathophysiological processes involved in the transition from fetal to neonatal life, pulmonary gas exchange, high-altitude pulmonary edema, and pulmonary hypertension. Although understanding of HPV has advanced significantly, major areas of ignorance and uncertainty await resolution.

A compendium of developmental gene expression in Lake Malawi cichlid fishes.

BACKGROUND: Lake Malawi cichlids represent one of a growing number of vertebrate models used to uncover the genetic and developmental basis of trait diversity. Rapid evolutionary radiation has resulted in species that share similar genomes but differ markedly in phenotypes including brains and behavior, nuptial coloration and the craniofacial skeleton. Research has begun to identify the genes, as well as the molecular and developmental pathways that underlie trait divergence. RESULTS: We assemble a compendium of gene expression for Lake Malawi cichlids, across pharyngula (the phylotypic stage) and larval stages of development, encompassing hundreds of gene transcripts. We chart patterns of expression in Bone morphogenetic protein (BMP), Fibroblast growth factor (FGF), Hedgehog (Hh), Notch and Wingless (Wnt) signaling pathways, as well as genes involved in neurogenesis, calcium and endocrine signaling, stem cell biology, and numerous homeobox (Hox) factors-in three planes using whole-mount in situ hybridization. Because of low sequence divergence across the Malawi cichlid assemblage, the probes we employ are broadly applicable in hundreds of species. We tabulate gene expression across general tissue domains, and highlight examples of unexpected expression patterns. CONCLUSIONS: On the heels of recently published genomes, this compendium of developmental gene expression in Lake Malawi cichlids provides a valuable resource for those interested in the relationship between evolution and development.

Digoxin inhibits development of hypoxic pulmonary hypertension in mice.

Chronic hypoxia is an inciting factor for the development of pulmonary arterial hypertension. The mechanisms involved in the development of hypoxic pulmonary hypertension (HPH) include hypoxia-inducible factor 1 (HIF-1)-dependent transactivation of genes controlling pulmonary arterial smooth muscle cell (PASMC) intracellular calcium concentration ([Ca(2+)](i)) and pH. Recently, digoxin was shown to inhibit HIF-1 transcriptional activity. In this study, we tested the hypothesis that digoxin could prevent and reverse the development of HPH. Mice were injected daily with saline or digoxin and exposed to room air or ambient hypoxia for 3 wk. Treatment with digoxin attenuated the development of right ventricle (RV) hypertrophy and prevented the pulmonary vascular remodeling and increases in PASMC [Ca(2+)](i), pH, and RV pressure that occur in mice exposed to chronic hypoxia. When started after pulmonary hypertension was established, digoxin attenuated the hypoxia-induced increases in RV pressure and PASMC pH and [Ca(2+)](i). These preclinical data support a role for HIF-1 inhibitors in the treatment of HPH.

Activation of hypoxia-inducible factor-1 in pulmonary arterial smooth muscle cells by endothelin-1.

Numerous cellular responses to hypoxia are mediated by the transcription factor hypoxia-inducible factor-1 (HIF-1). HIF-1 plays a central role in the pathogenesis of hypoxic pulmonary hypertension. Under certain conditions, HIF-1 may utilize feedforward mechanisms to amplify its activity. Since hypoxia increases endothelin-1 (ET-1) levels in the lung, we hypothesized that during moderate, prolonged hypoxia ET-1 might contribute to HIF-1 signaling in pulmonary arterial smooth muscle cells (PASMCs). Primary cultures of rat PASMCs were treated with ET-1 or exposed to moderate, prolonged hypoxia (4% O(2) for 60 h). Levels of the oxygen-sensitive HIF-1α subunit and expression of HIF target genes were increased in both hypoxic cells and cells treated with ET-1. Both hypoxia and ET-1 also increased HIF-1α mRNA expression and decreased mRNA and protein expression of prolyl hydroxylase 2 (PHD2), which is the protein responsible for targeting HIF-1α for O(2)-dependent degradation. The induction of HIF-1α by moderate, prolonged hypoxia was blocked by BQ-123, an antagonist of ET-1 receptor subtype A. The effects of ET-1 were mediated by increased intracellular calcium, generation of reactive oxygen species, and ERK1/2 activation. Neither ET-1 nor moderate hypoxia induced the expression of HIF-1α or HIF target genes in aortic smooth muscle cells. These results suggest that ET-1 induces a PASMC-specific increase in HIF-1α levels by upregulation of HIF-1α synthesis and downregulation of PHD2-mediated degradation, thereby amplifying the induction of HIF-1α in PASMCs during moderate, prolonged hypoxia.

Does Retiree Health Insurance Encourage Early Retirement?

The strong link between health insurance and employment in the United States may cause workers to delay retirement until they become eligible for Medicare at age 65. However, some employers extend health insurance benefits to their retirees, and individuals who are eligible for such retiree health benefits need not wait until age 65 to retire with group health coverage. We investigate the impact of retiree health insurance on early retirement using employee-level data from 54 diverse firms that are clients of Towers Watson, a leading benefits consulting firm. We find that retiree health coverage has its strongest effects at ages 62 through 64. Coverage that includes an employer contribution is associated with a 6.3 percentage point (36.2 percent) increase in the probability of turnover at age 62, a 7.7 percentage point (48.8 percent) increase in the probability of turnover at age 63, and a 5.5 percentage point (38.0 percent) increase in the probability of turnover at age 64. Conditional on working at age 57, such coverage reduces the expected retirement age by almost three months and reduces the total number of person-years worked between ages 58 and 64 by 5.6 percent.

Ca2+ responses of pulmonary arterial myocytes to acute hypoxia require release from ryanodine and inositol trisphosphate receptors in sarcoplasmic reticulum.

In pulmonary arterial smooth muscle cells (PASMC), acute hypoxia increases intracellular Ca(2+) concentration ([Ca(2+)](i)) by inducing Ca(2+) release from the sarcoplasmic reticulum (SR) and Ca(2+) influx through store- and voltage-operated Ca(2+) channels in sarcolemma. To evaluate the mechanisms of hypoxic Ca(2+) release, we measured [Ca(2+)](i) with fluorescent microscopy in primary cultures of rat distal PASMC. In cells perfused with Ca(2+)-free Krebs Ringer bicarbonate solution (KRBS), brief exposures to caffeine (30 mM) and norepinephrine (300 µM), which activate SR ryanodine and inositol trisphosphate receptors (RyR, IP(3)R), respectively, or 4% O(2) caused rapid transient increases in [Ca(2+)](i), indicating intracellular Ca(2+) release. Preexposure of these cells to caffeine, norepinephrine, or the SR Ca(2+)-ATPase inhibitor cyclopiazonic acid (CPA; 10 µM) blocked subsequent Ca(2+) release to caffeine, norepinephrine, and hypoxia. The RyR antagonist ryanodine (10 µM) blocked Ca(2+) release to caffeine and hypoxia but not norepinephrine. The IP(3)R antagonist xestospongin C (XeC, 0.1 µM) blocked Ca(2+) release to norepinephrine and hypoxia but not caffeine. In PASMC perfused with normal KRBS, acute hypoxia caused a sustained increase in [Ca(2+)](i) that was abolished by ryanodine or XeC. These results suggest that in rat distal PASMC 1) the initial increase in [Ca(2+)](i) induced by hypoxia, as well as the subsequent Ca(2+) influx that sustained this increase, required release of Ca(2+) from both RyR and IP(3)R, and 2) the SR Ca(2+) stores accessed by RyR, IP(3)R, and hypoxia functioned as a common store, which was replenished by a CPA-inhibitable Ca(2+)-ATPase.

Kinase-dependent activation of voltage-gated Ca2+ channels by ET-1 in pulmonary arterial myocytes during chronic hypoxia.

Exposure to chronic hypoxia (CH) causes pulmonary hypertension. The vasoconstrictor endothelin-1 (ET-1) is thought to play a role in the development of hypoxic pulmonary hypertension. In pulmonary arterial smooth muscle cells (PASMCs) from chronically hypoxic rats, ET-1 signaling is altered, with the ET-1-induced change in intracellular calcium concentration (Δ[Ca(2+)](i)) occurring through activation of voltage-dependent Ca(2+) channels (VDCC) even though ET-1-induced depolarization via inhibition of K(+) channels is lost. The mechanism underlying this response is unclear. We hypothesized that activation of VDCCs by ET-1 following CH might be mediated by protein kinase C (PKC) and/or Rho kinase, both of which have been shown to phosphorylate and activate VDCCs. To test this hypothesis, we examined the effects of PKC and Rho kinase inhibitors on the ET-1-induced Δ[Ca(2+)](i) in PASMCs from rats exposed to CH (10% O(2), 3 wk) using the Ca(2+)-sensitive dye fura 2-AM and fluorescent microscopy techniques. We found that staurosporine and GF109203X, inhibitors of PKC, and Y-27632 and HA 1077, Rho kinase inhibitors, reduced the ET-1-induced Δ[Ca(2+)](i) by >70%. Inhibition of tyrosine kinases (TKs) with genistein or tyrphostin A23, or combined inhibition of PKC, TKs, and Rho kinase, reduced the Δ[Ca(2+)](i) to a similar extent as inhibition of either PKC or Rho kinase alone. The ability of PKC or Rho kinase to activate VDCCs in our cells was verified using phorbol 12-myristate 13-acetate and GTP-γ-S. These results suggest that following CH, the ET-1-induced Δ[Ca(2+)](i) in PASMCs occurs via Ca(2+) influx through VDCCs mediated primarily by PKC, TKs, and Rho kinase.

Hypoxia-induced migration in pulmonary arterial smooth muscle cells requires calcium-dependent upregulation of aquaporin 1.

Pulmonary arterial smooth muscle cell (PASMC) migration is a key component of the vascular remodeling that occurs during the development of hypoxic pulmonary hypertension, although the mechanisms governing this phenomenon remain poorly understood. Aquaporin-1 (AQP1), an integral membrane water channel protein, has recently been shown to aid in migration of endothelial cells. Since AQP1 is expressed in certain types of vascular smooth muscle, we hypothesized that AQP1 would be expressed in PASMCs and would be required for migration in response to hypoxia. Using PCR and immunoblot techniques, we determined the expression of AQPs in pulmonary vascular smooth muscle and the effect of hypoxia on AQP levels, and we examined the role of AQP1 in hypoxia-induced migration in rat PASMCs using Transwell filter assays. Moreover, since the cytoplasmic tail of AQP1 contains a putative calcium binding site and an increase in intracellular calcium concentration ([Ca(2+)](i)) is a hallmark of hypoxic exposure in PASMCs, we also determined whether the responses were Ca(2+) dependent. Results were compared with those obtained in aortic smooth muscle cells (AoSMCs). We found that although AQP1 was abundant in both PASMCs and AoSMCs, hypoxia selectively increased AQP1 protein levels, [Ca(2+)](i), and migration in PASMCs. Blockade of Ca(2+) entry through voltage-dependent Ca(2+) or nonselective cation channels prevented the hypoxia-induced increase in PASMC [Ca(2+)](i), AQP1 levels, and migration. Silencing AQP1 via siRNA also prevented hypoxia-induced migration of PASMCs. Our results suggest that hypoxia induces a PASMC-specific increase in [Ca(2+)](i) that results in increased AQP1 protein levels and cell migration.

Enhancement of myofilament calcium sensitivity by acute hypoxia in rat distal pulmonary arteries.

Hypoxic contraction of pulmonary arterial smooth muscle is thought to require increases in both intracellular Ca(2+) concentration ([Ca(2+)](i)) and myofilament Ca(2+) sensitivity, which may or may not be endothelium-dependent. To examine the effects of hypoxia and endothelium on Ca(2+) sensitivity in pulmonary arterial smooth muscle, we measured the relation between [Ca(2+)](i) and isometric force at 37°C during normoxia (21% O(2)-5% CO(2)) and after 30 min of hypoxia (1% O(2)-5% CO(2)) in endothelium-intact (E+) and -denuded (E-) rat distal intrapulmonary arteries (IPA) permeabilized with staphylococcal α-toxin. Endothelial denudation enhanced Ca(2+) sensitivity during normoxia but did not alter the effects of hypoxia, which shifted the [Ca(2+)](i)-force relation to higher force in E+ and E- IPA. Neither hypoxia nor endothelial denudation altered Ca(2+) sensitivity in mesenteric arteries. In E+ and E- IPA, hypoxic enhancement of Ca(2+) sensitivity was abolished by the nitric oxide synthase inhibitor N(ω)-nitro-l-arginine methyl ester (30 µM), which shifted normoxic [Ca(2+)](i)-force relations to higher force. In E- IPA, the Rho kinase antagonist Y-27632 (10 µM) shifted the normoxic [Ca(2+)](i)-force relation to lower force but did not alter the effects of hypoxia. These results suggest that acute hypoxia enhanced myofilament Ca(2+) sensitivity in rat IPA by decreasing nitric oxide production and/or activity in smooth muscle, thereby revealing a high basal level of Ca(2+) sensitivity, due in part to Rho kinase, which otherwise did not contribute to Ca(2+) sensitization by hypoxia.

Acetazolamide prevents hypoxia-induced reactive oxygen species generation and calcium release in pulmonary arterial smooth muscle.

Upon sensing a reduction in local oxygen partial pressure, pulmonary vessels constrict, a phenomenon known as hypoxic pulmonary vasoconstriction. Excessive hypoxic pulmonary vasoconstriction can occur with ascent to high altitude and is a contributing factor to the development of high-altitude pulmonary edema. The carbonic anhydrase inhibitor, acetazolamide, attenuates hypoxic pulmonary vasoconstriction through stimulation of alveolar ventilation via modulation of acid-base homeostasis and by direct effects on pulmonary vascular smooth muscle. In pulmonary arterial smooth muscle cells (PASMCs), acetazolamide prevents hypoxia-induced increases in intracellular calcium concentration ([Ca2+]i), although the exact mechanism by which this occurs is unknown. In this study, we explored the effect of acetazolamide on various calcium-handling pathways in PASMCs. Using fluorescent microscopy, we tested whether acetazolamide directly inhibited store-operated calcium entry or calcium release from the sarcoplasmic reticulum, two well-documented sources of hypoxia-induced increases in [Ca2+]i in PASMCs. Acetazolamide had no effect on calcium entry stimulated by store-depletion, nor on calcium release from the sarcoplasmic reticulum induced by either phenylephrine to activate inositol triphosphate receptors or caffeine to activate ryanodine receptors. In contrast, acetazolamide completely prevented Ca2+-release from the sarcoplasmic reticulum induced by hypoxia (4% O2). Since these results suggest the acetazolamide interferes with a mechanism upstream of the inositol triphosphate and ryanodine receptors, we also determined whether acetazolamide might prevent hypoxia-induced changes in reactive oxygen species production. Using roGFP, a ratiometric reactive oxygen species-sensitive fluorescent probe, we found that hypoxia caused a significant increase in reactive oxygen species in PASMCs that was prevented by 100 µM acetazolamide. Together, these results suggest that acetazolamide prevents hypoxia-induced changes in [Ca2+]i by attenuating reactive oxygen species production and subsequent activation of Ca2+-release from sarcoplasmic reticulum stores.

Knockdown of stromal interaction molecule 1 attenuates store-operated Ca2+ entry and Ca2+ responses to acute hypoxia in pulmonary arterial smooth muscle.

Stromal interaction molecule 1 (STIM1) is a recently discovered membrane-spanning protein thought to sense lumenal Ca(2+) in sarco(endo)plasmic reticulum (SR/ER) and transduce activation of Ca(2+)-permeable store-operated channels (SOC) in plasmalemma in response to SR/ER Ca(2+) depletion. To evaluate the role of STIM1 and a closely related protein, STIM2, in Ca(2+) signaling of rat distal pulmonary arterial smooth muscle cells (PASMC) during hypoxia, we used fluorescent microscopy and the Ca(2+)-sensitive dye, fura 2, to measure basal intracellular Ca(2+) concentration ([Ca(2+)](i)), store-operated Ca(2+) entry (SOCE), and increases in [Ca(2+)](i) caused by acute hypoxia (4% O(2)) or depolarization (60 mmol/l KCl) in cells treated with small interfering RNA targeted to STIM1 (siSTIM1) or STIM2 (siSTIM2). As determined by real-time quantitative PCR analysis (qPCR), STIM1 mRNA was 200-fold more abundant than STIM2 in untreated control PASMC. siSTIM1 and siSTIM2 caused specific and significant knockdown of both mRNA measured by qPCR and protein measured by Western blotting. siSTIM1 markedly inhibited SOCE and abolished the sustained [Ca(2+)](i) response to hypoxia but did not alter the initial transient [Ca(2+)](i) response to hypoxia, the [Ca(2+)](i) response to depolarization, or basal [Ca(2+)](i). The only effect of siSTIM2 was a smaller inhibition of SOCE. We conclude that STIM1 was quantitatively more important than STIM2 in activation of SOC in rat distal PASMC and that the increase in [Ca(2+)](i) induced by acute hypoxia in these cells required SR Ca(2+) release and STIM1-dependent activation of SOC.

Human osteoarthritic chondrocytes are impaired in matrix metalloproteinase-13 inhibition by IFN-gamma due to reduced IFN-gamma receptor levels.

OBJECTIVE: Human osteoarthritic (OA) cartilage type-II collagen is preferentially cleaved by the proinflammatory cytokine-induced matrix metalloproteinases-13 (MMP-13). Interferon-gamma (IFN-gamma) potently inhibits interleukin-1 (IL-1)-induced MMP-13 expression in healthy chondrocytes. Our goal was to study the previously unknown impact of IFN-gamma on MMP-13 in OA and compare the levels and functional activity of IFN-gamma receptor (IFN-gammaR1) in healthy and OA chondrocytes. METHODS: Chondrocytes were obtained from OA patients and non-arthritic control subjects and treated with IL-1+ or- IFN-gamma. MMP-13 mRNA and protein expression were measured by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. IFN-gammaR1 expression was assessed by flow cytometry, immunoprecipitation and immunohistochemistry with fluorescein-labeled antibody. IFN-gammaR1 was neutralized with its antibody and signal transducer and activator of transcription 1 (STAT1) phosphorylation analyzed by Western blotting. OA chondrocytes were also transfected with control and IFN-gammaR1 expression vectors. RESULTS: OA chondrocytes displayed a drastically impaired MMP-13 suppression by IFN-gamma compared to control cells. IFN-gammaR1 levels were significantly decreased in OA chondrocytes as assessed by flow cytometry, immunoprecipitation and immunohistochemistry. Consequently, IFN-gamma-stimulated STAT1 phosphorylation mediated by IFN-gammaR1 was also considerably reduced in OA patient chondrocytes. IFN-gammaR1 overexpression in OA cells restored MMP-13 suppression by IFN-gamma. CONCLUSIONS: Ability of IFN-gamma to suppress IL-1-induced MMP-13 expression is diminished in OA chondrocytes due to decreased IFN-gammaR1 levels, activity and impaired downstream signal transduction. Therefore, IFN-gammaR1 modulation and weakened endogenous IFN-gamma response may be important mechanisms in OA pathogenesis and cartilage degradation.

Duchenne muscular dystrophy involving translocation of the dmd gene next to ribosomal RNA genes.

Duchenne muscular dystrophy (DMD) is a severe X-linked disorder leading to early death of affected males. Females with the disease are rare, but seven are known to be affected because of a chromosomal rearrangement involving a site at or near the dmd gene on the X chromosome. One of the seven has a translocation between the X and chromosome 21. The translocation-derived chromosomes from this patient have been isolated, and the translocation is shown to have split the block of genes encoding ribosomal RNA on the short arm of chromosome 21. Thus ribosomal RNA gene probes may be used to identify a junction fragment from the translocation site, allowing access to cloned segments of the X at or near the dmd gene and presenting a new approach to the study of this disease.

Molecular analysis of a constitutional X-autosome translocation in a female with muscular dystrophy.

The gene responsible for Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) maps to the X chromosome short arm, band Xp21. In a few females with DMD or BMD, the Xp21 region is disrupted by an X-autosome translocation. Accumulating evidence suggests that the exchange has physically disrupted the DMD/BMD locus to cause the disease. One affected female with a t(X;21)(p21;p12) translocation was studied in detail. The exchange points from both translocation chromosomes were cloned, restriction-mapped, and sequenced. The translocation is reciprocal, but not conservative. A small amount of DNA is missing from the translocated chromosomes; 71 to 72 base pairs from the X chromosome and 16 to 23 base pairs from the 28S ribosomal gene on chromosome 21.

Human ribosomal RNA genes: orientation of the tandem array and conservation of the 5' end.

The multiple copies of the human ribosomal RNA genes (rDNA) are arranged as tandem repeat clusters that map to the middle of the short arms of chromosomes 13, 14, 15, 21, and 22. Concerted evolution of the gene family is thought to be mediated by interchromosomal recombination between rDNA repeat units, but such events would also result in conservation of the sequences distal to the rDNA on these five pairs of chromosomes. To test this possibility, a DNA fragment spanning the junction between rDNA and distal flanking sequence has been cloned and characterized. Restriction maps, sequence data, and gene mapping studies demonstrate that (i) the rRNA genes are transcribed in a telomere-to-centromere direction, (ii) the 5' end of the cluster and the adjacent non-rDNA sequences are conserved on the five pairs of chromosomes, and (iii) the 5' end of the cluster is positioned about 3.7 kb upstream from the transcription initiation site of the first repeat unit. The data support a model of concerted evolution by interchromosomal recombination.

Duchenne muscular dystrophy gene expression in normal and diseased human muscle.

A probe for the 5' end of the Duchenne muscular dystrophy (DMD) gene was used to study expression of the gene in normal human muscle, myogenic cell cultures, and muscle from patients with DMD. Expression was found in RNA from normal fetal muscle, adult cardiac and skeletal muscle, and cultured muscle after myoblast fusion. In DMD muscle, expression of this portion of the gene was also revealed by in situ RNA hybridization, particularly in regenerating muscle fibers.

Investigating unsaturated fat, monensin, or bromoethanesulfonate in continuous cultures retaining ruminal protozoa. I. Fermentation, biohydrogenation, and microbial protein synthesis.

Methane is an end product of ruminal fermentation that is energetically wasteful and contributes to global climate change. Bromoethanesulfonate, animal-vegetable fat, and monensin were compared with a control treatment to suppress different functional groups of ruminal prokaryotes in the presence or absence of protozoa to evaluate changes in fermentation, digestibility, and microbial N outflow. Four dual-flow continuous culture fermenter systems were used in 4 periods in a 4 x 4 Latin square design split into 2 subperiods. In subperiod 1, a multistage filter system (50-microm smallest pore size) retained most protozoa. At the start of subperiod 2, conventional filters (300-microm pore size) were substituted to efflux protozoa via filtrate pumps over 3 d; after a further 7 d of adaptation, the fermenters were sampled for 3 d. Treatments were retained during both subperiods. Flow of total N and digestibilities of NDF and OM were 18, 16, and 9% higher, respectively, for the defaunated subperiod but were not different among treatments. Ammonia concentration was 33% higher in the faunated fermenters but was not affected by treatment. Defaunation increased the flow of nonammonia N and bacterial N from the fermenters. Protozoal counts were not different among treatments, but bromoethanesulfonate increased the generation time from 43.2 to 55.6 h. Methanogenesis was unaffected by defaunation but tended to be increased by unsaturated fat. Defaunation did not affect total volatile fatty acid production but decreased the acetate:propionate ratio; monensin increased production of isovalerate and valerate. Biohydrogenation of unsaturated fatty acids was impaired in the defaunated fermenters because effluent flows of oleic, linoleic, and linolenic acids were 60, 77, and 69% higher, and the ratio of vaccenic acid:unsaturated FA ratio was decreased by 34% in the effluent. This ratio was increased in both subperiods with the added fat diet, indicating an accumulation of intermediates of biohydrogenation. However, the flow of 18:2 conjugated linoleic acid was unaffected by defaunation or by treatments other than added fat. The flows of trans-10, trans-11, and total trans-18:1 fatty acids were not affected by monensin or faunation status.