Plasma Progesterone Assays in Superovulated Cattle

Plasma progesterone was measured in 14 normally cycling heifers and cows subjected to non-surgical recoveries of embryos. A radioimmunoassay (RIA) method was used for progesterone determination. The average progesterone concentration increased from 7.5 to 11.6 ng/ml in 8 of the animals following treatment with PMSG on day 8–12. Six animals had a decrease from 5.0 ± 2.1 to 3.9 ± 2.5 ng/ml. The overall increase was from 6.4 ± 2.7 ng/ml to 8.3 ± 4.8 ng/ ml. Prostaglandin F2a-analogue (cloprostenol) treatment resulted in a sharp decrease in plasma progesterone followed by a rapid increase to an average of 46.8 ng/ml on day 16. A high degree of variability in this peak value was observed, and it was not correlated with the number of corpora lutea. The superovulatory cycle was generally prolonged. The heat following the superovulatory treatment was silent, and a typical ovarian resting period was observed during which the progesterone concentration remained low and the ovaries small.     

Transient electric birefringence of T-even bacteriophages. II. T4B in the presence of tryptophan and T4D.

Measurements of electric birefringence, sedimentation velocity, and biological adsorption rate are used to study the properties of bacteriophage T4B in the presence of excess tryptophan. The adsorption rate determined in borate buffer pH 9 (at 25°C) increases from 0.003 × 10^?8 ml min^?1 (0.025 M) to 0.130 × 10^?8 ml min^?1 (0.150 M). The Kerr coefficient, rotational diffusion coefficient, and the sedimentation coefficient of the phage are also dependent on buffer concentration and reach plateau values above 0.12 M given by K_sp = ?(275 ± 18) × 10^?9 OD^?1 cm² statvolt^?2, D_25,w = 133 ± 4 sec^?1, and s_20,w = 818 ± 11 S. From a comparison of electric birefringence measurements of T4B and T4D it is concluded that T4D and T4B (in the presence of excess tryptophan) exhibit a similar hydrodynamic behavior. The change in physical parameters is solely due to a shift in fiber configuration. At high buffer concentrations the fibers make an angle of approximately 3π/4 with the sheath and the permanent dipole moment is about 200,000 D. This dipole moment is roughly ten times as large as that of a phage particle with nonextended fibers. This difference may be due to a change in hydrodynamic center upon fiber extension or to the presence of positive charges on the fiber tips, or both. At intermediate buffer concentrations the phage population behaves as if it were monodisperse. Probably not all six fibers are extended under such conditions.     

Embryo Transplantation in Cattle

Fourteen true repeat breeders with entirely normal oestrous cyclicity more than 1 year after calving and 14 control donor cows were superovulated with PMSG (2000 i.u.) and flushed non-surgically 6–8 days after the superovulatory heat. The superovulatory response was identical for the 2 groups such as assessed by the number of corpora lutea (9.4 ± 1.8 C.L. per repeat breeder and 9.1 ± 1.5 per control cow), occurrence of ovarian overstimulation (polycysts), presence of a non-countable amount of corpora lutea, negative outcome of the flushings and the number of recovered embryos (5.8 ± 1.0 embryos per repeat breeder and 6.0 ± 1.8 embryos per control cow). The most pronounced difference between the 2 categories of animals was related to the fertilization rate of embryos. In the repeat breeder group only 2.4 embryos per cow or 41 % were fertilized, whereas the control animals attained a fertilization rate of 4.9 embryos or 82 %. Since most factors liable to interfere with the fertilization process were identical for both groups (age, breed, nutritional and management conditions, semen quality, dose, AI-technician e.g.), it is believed that intraovarian, follicular, or follicular-dynamic conditions were responsible for producing a high proportion of non-fertilizable oocytes.     

Resonance Raman microspectroscopy of myeloperoxidase and cytochrome b558 in human neutrophilic granulocytes.

With (resonance) Raman microscospectroscopy, it is possible to investigate the chemical constitution of a very small volume (0.5 fl) in a living cell. We have measured resonance Raman spectra in the cytoplasm of living normal, myeloperoxidase (MPO)-deficient, and cytochrome b558-deficient neutrophils and in isolated specific and azurophilic granule fractions, using an excitation wavelength of 413.1 nm. Similar experiments were performed after reduction of the redox centers by the addition of sodium dithionite. The specific and azurophilic granules in both redox states appeared to have clearly distinguishable Raman spectra when exciting at a wavelength of 413.1 nm. The azurophilic granules and the cytochrome b558-deficient neutrophils showed Raman spectra similar to that of the isolated MPO. The spectra of the specific granules and the MPO-deficient neutrophils corresponded very well to published cytochrome b558 spectra. The resonance Raman spectrum of the cytoplasmic region of normal neutrophilic granulocytes could be fitted with a combination of the spectra of the specific and azurophilic granules, which shows that the Raman signal of neutrophilic granulocytes mainly originates from MPO and cytochrome b558, at an excitation wavelength of 413.1 nm.     

Simultaneous height and adhesion imaging of antibody-antigen interactions by atomic force microscopy.

Specific molecular recognition events, detected by atomic force microscopy (AFM), so far lack the detailed topographical information that is usually observed in AFM. We have modified our AFM such that, in combination with a recently developed method to measure antibody-antigen recognition on the single molecular level (Hinterdorfer, P., W. Baumgartner, H. J. Gruber, K. Schilcher, and H. Schindler, Proc. Natl. Acad. Sci. USA 93:3477-3481 (1996)), it allows imaging of a submonolayer of intercellular adhesion molecule-1 (ICAM-1) in adhesion mode. We demonstrate that for the first time the resolution of the topographical image in adhesion mode is only limited by tip convolution and thus comparable to tapping mode images. This is demonstrated by imaging of individual ICAM-1 antigens in both the tapping mode and the adhesion mode. The contrast in the adhesion image that was measured simultaneously with the topography is caused by recognition between individual antibody-antigen pairs. By comparing the high-resolution height image with the adhesion image, it is possible to show that specific molecular recognition is highly correlated with topography. The stability of the improved microscope enabled imaging with forces as low as 100 pN and ultrafast scan speed of 22 force curves per second. The analysis of force curves showed that reproducible unbinding events on subsequent scan lines could be measured.     

Optical tracking and detection of immunomagnetically selected and aligned cells

We have developed a platform for cell analysis based on immunomagnetic selection and magnetic alignment of cells in combination with an epi-illumination tracking and detection system. Whole blood was labeled with ferromagnetic nanoparticles and fluorescent probes, and placed in a magnetic field in a chamber. Cells labeled with ferromagnetic nanoparticles moved upward and aligned along ferromagnetic lines deposited by lithographic techniques on an optically transparent surface of the chamber. An epi-illumination system using a 635 nm laser diode as a light source scanned the lines and measured signals obtained from the aligned cells. The cell counts per unit of blood volume obtained with the system correlated well with those obtained from the counts from a standard hematology analyzer and flow cytometer. The cell analysis platform is significantly less complex and more sensitive than current cell analysis equipment and provides additional functionality through its ability to subject the cells to repeated and varied analyses while they remain in a natural environment (i.e., whole blood).     

Estrus characterization in superovulated cattle

Based on frequent examinations per rectum and on external signs of estrus, the intensity of estrus was characterized in 107 superovulated dairy cows and heifers. Three intensity groups were defined: strong (n=57), moderate (n=34), and weak (n=16). Frequent blood samples were obtained for analyses of estradiol-17β and LH. The ovaries were exposed through surgery at various intervals after the time of prostaglandin administration, and ovarian findings (follicles, premature ovulations) were described. A total of 527 oocytes was recovered from 998 follicles aspirated, and, of these, 420 were processed for chromosomal evaluation of their nuclear maturational stage.

The weak estrus intensity group differed from the strong and moderate groups in several ways. Cows and heifers in the weak intensity group had more frequent deviations in plasma hormonal patterns, a higher proportion of animals with only a few follicles, and more animals exhibiting premature ovulations. The mean oocyte recovery rate was lower in the weak group as compared with that of the other 2 groups, since more oocytes could not be accounted for in this group during the evaluation process, indicating a higher degree of oocyte fragility. Furthermore, a larger number of oocytes in the weak group displayed no identifiable chromosomes, than in the remaining 2 groups. However, apparently normal oocytes were also found in the weak group.

It is concluded that accurate characterization of estrus in superovulated cattle can be a valuable tool for identifying donors with a high risk for inadequate oocyte maturation, which in turn could lead to inferior embryo yield.     

Formation of rhinovirus-soluble ICAM-1 complexes and conformational changes in the virion.

Viral receptors serve both to target viruses to specific cell types and to actively promote the entry of bound virus into cells. Human rhinoviruses (HRVs) can form complexes in vitro with a truncated soluble form of the HRV cell surface receptor, ICAM-1. These complexes appear to be stoichiometric, with approximately 60 ICAM molecules bound per virion or 1 ICAM-1 molecule per icosahedral face of the capsid. The complex can have two fates, either dissociating to yield free virus and free ICAM-1 or uncoating to break down to an 80S empty capsid which has released VP4, viral RNA, and ICAM-1. This uncoating in vitro mimics the uncoating of virus during infection of cells. The stability of the virus-receptor complex is dependent on temperature and the rhinovirus serotype. HRV serotype 14 (HRV14)-ICAM-1 complexes rapidly uncoat, HRV16 forms a stable virus-ICAM complex which does not uncoat detectably at 34 degrees C, and HRV3 has an intermediate phenotype. Rhinovirus can also uncoat after exposure to mildly acidic pH. The sensitivities of individual rhinovirus serotypes to ICAM-1-mediated virus uncoating do not correlate with uncoating promoted by incubation at low pH, suggesting that these two means of virus destabilization occur by different mechanisms. Soluble ICAM-1 and low pH do not act synergistically to promote uncoating. The rate of uncoating does appear to be inversely related to virus affinity for its receptor.     

The “Trojan horse” strategy: Seed fungal endophyte symbiosis helps to explain the invasion success of the grass,Poa annua,in Maritime Antarctica

Aim

Poa annua L. (annual bluegrass) is presently the sole invasive vascular plant species to have successfully established in Maritime Antarctica, where it poses a significant conservation threat to native plant species. However, the reasons for its success in the region have yet to be established. Here, we determined whether the invasiveness of P. annua, and its competitiveness with the native Antarctic hairgrass Deschampsia antarctica, is influenced by symbioses formed with seed fungal endophytes, and whether plants derived from seeds from four global regions differ in their performance.

Locations

Four regions (Maritime Antarctica, sub-Antarctica, South America and Europe).

Methods

Endophyte frequency was measured in P. annua seeds collected from the four regions. The germination, survival, biomass accumulation, flowering and competitiveness with D. antarctica of P. annua plants grown from endophyte-uncolonised and uncolonised seeds was determined in the laboratory. The effects of endophytes on P. annua seed germination and survival and seedling osmoprotection were also assessed in the Maritime Antarctic natural environment using locally-sourced seeds.

Results

Endophytes were at least twice as frequent in seeds from Maritime Antarctica than in those from other regions. A higher proportion of endophyte-colonized seeds germinated and survived than did uncolonised seeds, but only when they originated from Maritime Antarctica. Seed endophytes increased the competitiveness of P. annua with D. antarctica, but only for plants grown from Maritime Antarctic seeds. In the field, endophyte-colonized seeds from Maritime Antarctica germinated and survived more frequently than uncolonised seeds, and osmoprotection was higher in seedlings grown from colonized seed.

Main Conclusions

The findings indicate beneficial effects of seed endophytes on invasion-related traits of P. annua, such as survival, germination success and flowering. Together with vegetative and reproductive traits facilitating the colonization process, the seed-fungal endophyte symbiosis can be invoked as an important factor explaining the invasiveness of P. annua in Maritime Antarctica.     

Associations between beta-tubulin and mitochondria in adult isolated heart myocytes as shown by immunofluorescence and immunoelectron microscopy

Summary We have investigated the associations between -tubulin and mitochondria in freshly isolated cardiac myocytes from the rat. Beta-tubulin was identified by using monoclonal antibodies for immunofluorescence and high resolution immunogold electron microscopy. In addition, conventional transmission and scanning electron microscopic studies were performed. After chemical stabilization in a formaldehyde solution, the myocytes were shock-frozen at –150°C, cryosectioned at –70°C and subsequently processed for immunohistochemical and immunocytochemical microscopy. A characteristic of the rod shaped myocytes is the presence of a dense network of microtubules in the cytoplasm displaying a pattern of strong anti--tubulin reaction. The complexity of this network however varies considerably among the myocytes reflecting microtubule dynamic instability. Further, our findings demonstrate that the -tubulin label in rod cells is confined to the perinuclear and interfibrillar spaces and, therefore, is largely colocalized with the cytoplasmic organelles. In myocytes undergoing severe contracture the distribution of -tubulin is entirely restricted to the outer mitochondrial-containing domain. This implies that, in a cell model with marked segregation of the contractile filaments and organelles, mitochondria are codistributed with microtubules in the total absence of desmin intermediate filaments. Moreover, our immunogold preparations demonstrate anti--tubulin labelling in the outer mitochondrial membrane as well as of fibres in close apposition to this membrane. These results indicate the presence of a specific -tubulin binding to the outer mitochondrial membrane that probably also involves microtubule based translocators and/or MAPs.