Fertility in seasonal-calving pasture-based lactating dairy cows following timed artificial insemination or timed embryo transfer with fresh or frozen in vitro-produced embryos.

The objective was to compare pregnancy per service event (P/S) in lactating dairy cows following timed artificial insemination (AI) or timed embryo transfer (ET) using either fresh or frozen in vitro-produced embryos. Oocytes were collected once per week for up to 9 wk using transvaginal ovum pick-up from elite dairy donors (ET-DAIRY; n = 40; Holstein-Friesian and Jersey) and elite beef donors (ET-ELITE-BEEF; n = 21; Angus). Both ET-DAIRY and ET-ELITE-BEEF donors consisted of heifers and cows. In addition, oocytes were collected from the ovaries of beef heifers of known pedigree following slaughter at a commercial abattoir (ET-COMM-BEEF; n = 119). Following in vitro maturation and fertilization, presumptive zygotes were cultured in vitro to the blastocyst stage. Grade 1 blastocysts were either transferred fresh or frozen for on-farm thawing and direct transfer. A total of 1,106 recipient cows (all lactating, predominantly Holstein-Friesian) located on 16 herdlets were blocked based on parity, calving date, and Economic Breeding Index, and randomly assigned to receive AI (n = 243) or ET (n = 863) after estrous synchronization with a 10-d Progesterone-synch protocol. Cows assigned to ET were further randomized to receive fresh (n = 187) or frozen (n = 178) ET-ELITE-BEEF embryos, fresh (n = 169) or frozen (n = 162) ET-DAIRY embryos, or fresh (n = 80) or frozen (n = 87) ET-COMM-BEEF embryos. Pregnancy was diagnosed using transrectal ultrasound on d 32 to 35 after synchronized ovulation and confirmed on d 62 to 65, at which time fetal sex was determined. Pregnancy per service event at d 32 was not different between AI (48.8%) and ET (48.9%) and did not differ between dairy and beef embryos (50.3% vs. 48.1%, respectively). However, P/S was less on d 32 following transfer of frozen embryos (41.6%) compared with fresh embryos (56.1%). Pregnancy loss between d 32 and 62 was greater for ET (15.1%) compared with AI (4.7%), with greater losses observed for frozen beef (18.5%), fresh beef (17.3%), and frozen dairy (19.2%) compared with fresh dairy (6.0%) embryos. Serum progesterone (P4) concentration on d 7 was associated with P/S at d 32 and 62. Cows in the quartile with the least serum P4 concentrations (quartile 1) had less probability of being pregnant on d 32 (33.4%) compared with cows in the 3 upper quartiles for serum P4 (45.7%, 55.6%, and 61.2% for quartile 2, quartile 3, and quartile 4, respectively). Sex ratio (male:female) at d 62 was skewed toward more male fetuses following ET (61.1:38.9) compared with AI (43.2:56.8) and was consistent with the sex ratio among in vitro blastocysts (61.2:38.8). In conclusion, P/S was similar for AI and ET, although pregnancy loss between d 32 and 62 was greater for ET than for AI.

A reexamination of the effects of creatine on muscle protein synthesis in tissue culture.

Experiments designed to test the hypothesis that intracellular creatine level regulates the synthesis of muscle specific proteins have failed to demonstrate any creatine regulatory effect. Manipulation of the extracellular creatine in culture medium over a 5,700-fold range (1.3-7.4 mM) was successful in altering intracellular total creatine by only a factor of 20 (1.4-42 mg creatine/mg protein), an indication that muscle cells are able to regulate intracellular creatine levels over a wide range of external creatine concentrations. Alterations of cell creatine had no effect on either total protein synthesis or synthesis of myosin heavy chain. Methods were perfected to measure total creatine, and incorporation of [3H]leucine into total protein and purified myosin heavy chain from the same culture dish to avoid the possibility of variation between dishes. The creatine analog 1-carboxymethyl-2-iminohexahydropyrimidine (CMIP) previously reported to stimulate myosin synthesis in culture was found to depress creatine accumulation by cells and depressed total protein synthesis and synthesis of myosin heavy chain. This inhibitory action of CMIP is consistent with the reported competitive inhibition of creatine kinase and presumed interference with energy metabolism.

Specificity of creatine in the control of muscle protein synthesis.

This study provides additional evidence that creatine, an end product of contraction unique to muscle, is involved in the control of muscle protein synthesis. Creatine is shown to stimulate selectively the rate of synthesis of two major contractile proteins, actin and myosin heavy chain, in cultures of differentiating skeletal muscle. Creatine affects only the rate of synthesis and not the rate of degradation. Several creatine analogs are as effective as creatine in stimulating muscle protein synthesis, creatinine and amino acids such as arginine and glycine are not. Creatine stimulates myosin heavy chain synthesis twofold in cultures of embryonic muscle grown in either normal or dialyzed media.

Low Altitude Solar Magnetic Reconnection, Type III Solar Radio Bursts, and X-ray Emissions.

Type III solar radio bursts are the Sun's most intense and frequent nonthermal radio emissions. They involve two critical problems in astrophysics, plasma physics, and space physics: how collective processes produce nonthermal radiation and how magnetic reconnection occurs and changes magnetic energy into kinetic energy. Here magnetic reconnection events are identified definitively in Solar Dynamics Observatory UV-EUV data, with strong upward and downward pairs of jets, current sheets, and cusp-like geometries on top of time-varying magnetic loops, and strong outflows along pairs of open magnetic field lines. Type III bursts imaged by the Murchison Widefield Array and detected by the Learmonth radiospectrograph and STEREO B spacecraft are demonstrated to be in very good temporal and spatial coincidence with specific reconnection events and with bursts of X-rays detected by the RHESSI spacecraft. The reconnection sites are low, near heights of 5-10 Mm. These images and event timings provide the long-desired direct evidence that semi-relativistic electrons energized in magnetic reconnection regions produce type III radio bursts. Not all the observed reconnection events produce X-ray events or coronal or interplanetary type III bursts; thus different special conditions exist for electrons leaving reconnection regions to produce observable radio, EUV, UV, and X-ray bursts.

Polarization of tryptophan fluorescence from single striated muscle fibers. A molecular probe of contractile state.

Instrumentation has been developed to detect rapidly the polarization of tryptophan fluorescence from single muscle fibers in rigor, relaxation, and contraction. The polarization parameter (P( perpendicular)) obtained by exiciting the muscle tryptophans with light polarized perpendicular to the long axis of the muscle fiber had a magnitude P( perpendicular) (relaxation) > P( perpendicular) (contraction) > P( perpendicular) (rigor) for the three types of muscle fibers examined (glycerinated rabbit psoas, glycerinated dorsal longitudinal flight muscle of Lethocerus americanus, and live semitendinosus of Rana pipiens). P( perpendicular) from single psoas fibers in rigor was found to increase as the sarcomere length increased but in relaxed fibers P( perpendicular) was independent of sarcomere length. After rigor, pyrophosphate produced little or no change in P( perpendicular), but following an adenosine triphosphate (ATP)-containing solution, pyrophosphate produced a value of P( perpendicular) that fell between the contraction and relaxation values. Sinusoidal or square wave oscillations of the muscle of amplitude 0.5-2.0% of the sarcomere length and frequency 1, 2, or 5 Hz were applied in rigor when the myosin cross-bridges are considered to be firmly attached to the thin filaments. No significant changes in P( perpendicular) were observed in either rigor or relaxation. The preceding results together with our present knowledge of tryptophan distribution in the contractile proteins has led us to the conclusion that the parameter P( perpendicular) is a probe of the contractile state of myosin which is probably sensitive to the orientation of the myosin S1 subfragment.

Nucleotide regulation of vasoactive intestinal peptide binding to bovine thyroid plasma membranes.

The specific binding of vasoactive intestinal peptide (VIP) to bovine thyroid plasma membranes is inhibited by guanine nucleotides. Guanosine 5'-triphosphate (GTP) and the non-hydrolyzable GTP analogs guanosine 5'-beta,gamma-imidotriphosphate (Gpp(NH)p) and guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S) inhibited markedly the binding of VIP to its receptors. This inhibition was higher with GTP than with Gpp(NH)p and GTP-gamma-S and was due to an increase of the rate of dissociation of peptide bound to membranes. Other nucleotides did not show any effect.

The micromechanics of contraction.

This paper suggests the molecular mechanism in muscle whereby some of the delta G of ATP hydrolysis is converted to mechanical work. There is good evidence for thinking that all mechanical, thermal, and chemical observations of muscle result from the summated, repetitive, operation of identical unitary "engines". Such an engine is the S-1 moiety of a myosin crossbridge and the adjacent actin. The operational principle is evident on realizing that S-1 is a particle with two interactive ligand (ATP and actin) binding sites. The change resulting from binding nucleotide is propagated to the actin-binding site, there specifying affinity and angles of attachment. Repetition follows because stepwise enzymatic degradation through intermediates is equivalent to cyclical changing of the site occupant. Trans- (S-1) propagation ensures a work cycle because actin held at a succession of relative positions can generate external work. Proximity mapping and protein chemical studies of S-1 suggest that variation in the position at which actin is held results from the simultaneous operation of a continuing (S-1)-actin contact and a polyphosphate charge-modulated coulombic contact.

The region in myosin S-1 that may be involved in energy transduction.

Newly-reported structural information about certain proximities between points on bound nucleotide and points on the heavy chain of myosin S-1 are incorporated into a previously-reported [Botts, J. Thomason, J.F. & Morales, M.F. Proc. Nat. Acad. Sci. USA, 86, 2204-2208 (1989)] structure of S-1. The resulting, enhanced structure is then used to identify some functionalities (e.g., the ATP-perturbable tryptophans), and to explain certain observations (e.g., some concerning the role of bound Mg2+ in the spectral response of TNBS-labelled Lys-83, and some concerning the response of the S-1 CD signal to nucleotide binding and to temperature change). These considerations lead to the suggestion that a strand of the 50 kDa "domain" (residues 510 to 540), and a strand of the 20 kDa 'domain' (residues 697-719) are involved in transmitting the effects of nucleotide binding and hydrolysis to the loop (constituted from the same "domain") that reaches a major (S-1)-actin interface.

Smooth muscle myosin. Amino acid residues responsible for the hydrolysis of ATP.

From their crystallographic comparisons, Fisher et al. (Biochemistry 34, 8960-8972, 1995) have proposed that in an important transition of myosin heads (M), M.ATP-->M.ADP.Pi, an interdomain rotation occurs in Gly468 (of chicken smooth muscle myosin) and that the rotated state is stabilized by newly-formed interdomain contacts including the salt link between Glu470 and Arg247 (of chicken smooth muscle myosin). Here, we have studied the effects of Gly468, Glu470, and Arg247 mutations on the hydrolysis of ATP. The G468A HMM did not show a significant ATPase activity, a stoichiometric initial phosphate burst, and tryptophan fluorescence enhancement attributed to bound ADP.Pi. The E470A HMM also did not show a significant ATPase activity and the phosphate burst, but the mutant gave tryptophan response attributed to bound ATP. The E470R/R247E HMM exhibited an ATPase activity and the phosphate burst which were comparable to those of the wild-type HMM, whereas neither the E470R HMM nor the R247E HMM showed such a significant ATPase activity and burst. We thus propose that both an unhindered rotation and a salt link that stabilizes the rotated state are necessary for ATP hydrolysis.

Evidence for TeV gamma-ray emission from a region of the galactic plane.

Gamma-ray emission from a narrow band at the galactic equator has previously been detected up to 30 GeV. We report evidence for a TeV gamma-ray signal from a region of the galactic plane by Milagro, a large-field-of-view water Cherenkov detector for extensive air showers. An excess with a significance of 4.5 standard deviations has been observed from the region of galactic longitude l E (40 degrees, 100 degrees) and latitude /b/ < 5 degrees. Under the assumption of a simple power law spectrum, with no cutoff in the EGRET-Milagro energy range, the measured integral flux is phi gamma(>3.5 TeV) = (6.4 +/- 1.4 +/- 2.1) x 10(-11) cm(-2) s(-1) sr(-1). This flux is consistent with an extrapolation of the EGRET spectrum between 1 and 30 GeV in this galactic region.

Two kinds of transducer.

After recognizing that there are two kinds of biotransducers, attention is focused on the kind that transduces energy, as in muscle or in ion pumps. Using muscle as the example, it is suggested that the essential elements in the transducer (S-1 segment of myosin) are (1) An ATPase site that, in time, is occupied by a succesion of ligands ("intermediates"); (2) A spatially separate site for binding a second ligand (actin, in the case of muscle) that can be held in a variety of spatial relations; and (3) A transmitting mechanism (electric field, propagated structural distortion) that allows the conformation impressed by the ligand of (1) to be conducted over space and impose a specific relation to the ligand of (2).

Inferences from the equations used for describing the chemistry of contraction.

Schemes describing the chemical kinetic behavior of actin/myosin/ATP (i.e., contractile) systems are often describable as circles of isomerizations. In this paper explicit formulas for the steady-state concentrations of intermediate complexes and of the net reaction rate are given, and then the power spectrum of the concentration fluctuations is derived explicitly for certain cases, in a manner applicable to experiments.

Calculation of the polarized fluorescence from a labeled muscle fiber.

Equations are derived that explicitly relate fluorescence polarization observables on a labeled muscle fiber to attitude of the cross-bridges and to attitude of the labels within the cross-bridges.

Modifying preselected sites on proteins: the stretch of residues 633-642 of the myosin heavy chain is part of the actin-binding site.

We have designed an "antipeptide" capable of firmly and specifically interacting with a preselected stretch of myosin S-1 heavy chain. Covalent attachment of this antipeptide to its target stretch, residues 633-642, does not affect the intrinsic ATPase activities of the protein but significantly reduces the actin-binding capabilities of the myosin head.