Development and cellular organization ofPinus sylvesfris pollen tubes

The organization ofPinus sylvestris pollen tubes during growth was studied by video microscopy of living cells and by electron microscopy after freeze-fixation and freeze-substitution (FF-FS). Pollen germinated and the tubes grew slowly for a total period of about 7 days. Some of the grains formed two tubes, while 10–50% of the tubes ramified. These features are in accordance with development in vivo. The cytoplasmic hyaline cap at the tip disappeared during the 2nd or 3rd day of culture. Aggregates of starch grains progressively migrated from the grain into the tube and later into the branches. Vacuoles first appeared at day 2 and eventually filled large parts of the tube. The tube nucleus was located at variable distances from the tip. Some of the organelles showed linear movements in a mostly circulatory pattern, but the majority of the organelles showed brownian-like movements. Rhodamine-phalloidin-stained actin filaments had a gross axial orientation and were found throughout the tube including at the tip. The ultrastructure of pollen tubes was well preserved after FF-FS, but signs of shrinkage were visible. The secretory vesicles in growing tips were not organized in a vesicle cone, and coated pits had a low density with only local accumulations, which is in accordance with slow growth. The mitochondria contained small cristae and a darkly stained matrix and were located more towards the periphery of the tube, indicating low respiratory activity and low oxygen levels. The dictyosomes carried typical trans-Golgi networks, but some contained less than the normal number of cisternae. Other elements of the cytoplasm were irregularly spaced rough endoplasmic reticulum, many multivesicular bodies, lipid droplets and two types of vacuoles. The typical organization associated with tip growth in angiosperm pollen tubes, e.g.Nicotiana tabacum, was not present inP. sylvestris pollen tubes. The different morphology may relate to the growth rate and not to the type of growth.     

Gram staining apparatus for space station applications.

A self-contained, portable Gram staining apparatus (GSA) has been developed for use in the microgravity environment on board the Space Station Freedom. Accuracy and reproducibility of this apparatus compared with the conventional Gram staining method were evaluated by using gram-negative and gram-positive controls and different species of bacteria grown in pure cultures. A subsequent study was designed to assess the performance of the GSA with actual specimens. A set of 60 human and environmental specimens was evaluated with the GSA and the conventional Gram staining procedure. Data obtained from these studies indicated that the GSA will provide the Gram staining capability needed for the microgravity environment of space.     

M. tuberculosis and M. leprae translocate from the phagolysosome to the cytosol in myeloid cells

M. tuberculosis and M. leprae are considered to be prototypical intracellular pathogens that have evolved strategies to enable growth in the intracellular phagosomes. In contrast, we show that lysosomes rapidly fuse with the virulent M. tuberculosis- and M. leprae-containing phagosomes of human monocyte-derived dendritic cells and macrophages. After 2 days, M. tuberculosis progressively translocates from phagolysosomes into the cytosol in nonapoptotic cells. Cytosolic entry is also observed for M. leprae but not for vaccine strains such as M. bovis BCG or in heat-killed mycobacteria and is dependent upon secretion of the mycobacterial gene products CFP-10 and ESAT-6. The cytosolic bacterial localization and replication are pathogenic features of virulent mycobacteria, causing significant cell death within a week. This may also reveal a mechanism for MHC-based antigen presentation that is lacking in current vaccine strains.     

Ultrasonographic image attributes of non-ovulatory follicles and follicles with different luteal outcomes in gonadotropin-releasing hormone (GnRH)-treated anestrous ewes

Ultrasonographic images are composed of multiple square picture elements called pixels. Quantitative changes in numerical pixel values (echotexture) determined by computer-assisted analysis of digital images reflect discrete changes in the microscopic structure and physiological status of ovarian antral follicles. The objective of the present study was to determine and compare the ultrasonographic attributes of non-ovulatory antral follicles that grew to an ostensibly ovulatory diameter (> or =5mm) and follicles with different luteal outcomes in response to gonadotropin-releasing hormone (GnRH) in anestrous Western White Face ewes (n=34). All animals received GnRH injections (250ng i.v. every 2h for 24h) followed by a bolus injection of 125microg of GnRH i.v. Ovarian images obtained by repeated transrectal ultrasonography were digitized and subjected to computerized analyses to determine the changes in follicular size and echotexture of the follicular antrum and wall. At the beginning of GnRH treatment, follicles that formed inadequate corpora lutea following ovulation (ICL; n=22) had higher (P<0.001) pixel intensity of the central and peripheral antrum compared with non-ovulatory follicles (n=40). Pixel intensity of the central follicular antrum was greater (P<0.01) in follicles that formed ICL compared with follicles that formed normal (full-lifespan) CL post-treatment (NCL; n=20) and mean pixel heterogeneity of the follicular wall was greater (P<0.05) in non-ovulatory follicles compared with follicles that gave rise to NCL. At the time of GnRH bolus injection (i.e., induction of a synchronous LH surge), the mean diameter of non-ovulatory follicles was greater (P<0.01) than that of all ovulating follicles, and pixel heterogeneity of the central follicular antrum was lowest (P<0.05) in non-ovulatory follicles. The mean diameter of luteinized unovulated follicles (n=9) tended to be greater (P<0.10) at 2.5 and 3 days after emergence, and pixel intensity of the follicular wall was lower (P<0.05) compared with non-luteinized follicles (n=8) at 1.5 and 2.5 days after emergence (beginning of the growth from approximately 3mm onwards). In conclusion, ovarian antral follicles with different outcomes after GnRH treatment (in seasonally anestrous ewes) had distinctive ultrasonographic characteristics.     

The stoichiometry of antibody-mediated neutralization and enhancement of West Nile virus infection

Antibody binding to the icosahedral arrangement of envelope proteins on the surface of flaviviruses can result in neutralization or enhancement of infection. We evaluated how many antibodies must bind to a given epitope on West Nile virus (WNV) to achieve neutralization. The most potent monoclonal antibodies (mAbs) block infection at concentrations that result in low occupancy of accessible sites on the virion, with neutralization occurring when as few as 30 of 180 envelope proteins are bound. In contrast, weakly neutralizing mAbs recognize fewer sites on the virion and require almost complete occupancy to inhibit WNV infection. For all mAbs studied, enhancement of infection is possible in cells bearing activating Fc-gamma receptors when the number of mAbs docked to the virion is not sufficient for neutralization. Thus, neutralization is best described by a model requiring "multiple hits" with the cumulative functional outcome determined by interplay between antibody affinity and epitope accessibility.     

Comparative study of biosynthetic human growth hormone immunogenicity in growth hormone deficient children

The immunogenicities of six recombinant human growth hormone (rhGH) preparations, from KABI (A rhGH191 and B rhGH192), Eli Lilly (C), Nordisk (D), Sanofi (E) and Serono (F), used to treat 260 GH-deficient children, have been compared using a common specific and sensitive procedure for antibody determination. For this purpose we developed two immunoassays: a competitive liquid radioimmunoassay using 125I-rhGH, and an immunometric solid enzymoimmunoassay in which the rhGHs were immobilized. Blood samples were collected from the GH-deficient children before treatment and after 3, 6, 9, 12, 18 and 24 months of therapy. Human GH antibodies were detected in children treated with 3 of the 6 rhGH preparations. Seven percent of the patients treated with hormone A, 14% with hormone B and 22% with hormone C formed antibodies against the respective rhGH. Differences in capacity and affinity of the hGH antibodies were observed between these anti-GH-positive groups. They could be divided into 2 groups according to their immunopotency. One group (7, 14 and 6% of the patients treated with hormones A, B and C, respectively) developed anti-hGH antibodies with very low binding capacities (30-100 fmol/ml). The other group (16% of the patients treated with hormone C) developed IgG-type antibodies to hGH with higher binding capacities (200-1,200 fmol/ml) and a measurable binding affinity (Ka = 10(8) M-1). These hGH antibodies partially inhibited the binding of labeled GH to its specific liver membrane receptor. However, because of their low titer, they did not inhibit growth in the treated children.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of body size and gender on control of ventilation

Hypoxic (HVR) and hypercapnic (HCVR) ventilatory responses are influenced by both metabolic activity and hormonal factors. By studying 67 subjects of both sexes, including those at the extremes of stature, we examined the influence of gender, CO2 production (VCO2), O2 consumption (VO2), body surface area (BSA), and vital capacity (VC) on resting ventilation (VE), HVR, and HCVR. We measured resting VE, VO2, and VCO2 and then performed isocapnic progressive hypoxic and hypercapnic ventilatory responses. The effect of stature was reflected in higher VE and metabolic rate (both P less than 0.001) in tall men compared with short men that was ablated by correction for BSA. Perhaps because their heights vary less than those of the men, tall women were not statistically distinguishable from short women in any of these measured parameters. Tall men tended to have greater hypoxic chemosensitivity than short men but this was not significantly different (P = 0.07). Gender affected the control of ventilation in a number of ways. Men had higher VE (P less than 0.05) and metabolic rate (P less than 0.001) than women. Even after correction for BSA men still had higher metabolic rates. Women had higher VE/VCO2 than men (P less than 0.05) and lower resting end-tidal Pco2 (PETCO2) values (P less than 0.05). Both A, the shape parameter of the hyperbolic HVR curve, and HVR determined from mouth occlusion pressure (AP) were greater in women than in men, although only AP reached statistical significance. However, corrections of A for BSA (P less than 0.05), VCO2 (P less than 0.01), and VC (P less than 0.001) amplified these differences.(ABSTRACT TRUNCATED AT 250 WORDS)