Formaldehyde in cryoprotectant propanediol and effect on mouse zygotes

Cryopreservation of human zygotes and embryos has been routinely performed by in-vitro fertilization clinics for many years. Karran and Legge (1996) first reported that formaldehyde (FA) present in the cryoprotective solutions can have a deleterious effect on mouse oocytes. FA is a cytotoxic, carcinogenic and mutagenic chemical. The effect of FA on mouse zygotes was investigated. In addition, the concentrations of FA in propanediol (PROH) obtained from various sources were determined. Pooled 1-cell embryos were dispensed into droplets of modified Ham's F10 or human tubal fluid containing various concentrations of FA. Since bovine serum albumin (BSA) may minimize toxicity additional trials were done as above in the absence of BSA. FA concentration in the standard 1.5 M PROH, from different sources in water, was measured in the same assay using a standard curve of 0-100 microM FA. FA in a complex medium had a significant deleterious effect on embryo development and hatching but only at 1 mM concentration (P < 0.000001; see Tables I-III). There was no significant effect of FA at 100 microM. However, in a simple medium even 50 microM FA decreased embryo hatching. FA was present in 1.5 M PROH from different sources (range 1.0-35.3 microM concentration). It appears that FA concentrations do not increase with storage because FA concentrations were low even after opening and storage for 3 years on the shelf. This suggests that FA is a contaminant during the manufacturing process and may vary from manufacturer to manufacturer and batch to batch. Until further studies are done to confirm the lack of toxicity to embryos during cryopreservation (with or without FA scavengers) it may be prudent to screen all batches of cryoprotectants for FA as part of quality control.      

On the noise variance of a digital mammography system

A recent paper by Cooper et al. [Med. Phys. 30, 2614-2621 (2003)] contains some apparently anomalous results concerning the relationship between pixel variance and x-ray exposure for a digital mammography system. They found an unexpected peak in a display domain pixel variance plot as a function of 1/mAs (their Fig. 5) with a decrease in the range corresponding to high display data values, corresponding to low x-ray exposures. As they pointed out, if the detector response is linear in exposure and the transformation from raw to display data scales is logarithmic, then pixel variance should be a monotonically increasing function in the figure. They concluded that the total system transfer curve, between input exposure and display image data values, is not logarithmic over the full exposure range. They separated data analysis into two regions and plotted the logarithm of display image pixel variance as a function of the logarithm of the mAs used to produce the phantom images. They found a slope of minus one for high mAs values and concluded that the transfer function is logarithmic in this region. They found a slope of 0.6 for the low mAs region and concluded that the transfer curve was neither linear nor logarithmic for low exposure values. It is known that the digital mammography system investigated by Cooper et al. has a linear relationship between exposure and raw data values [Vedantham et al., Med. Phys. 27, 558-567 (2000)]. The purpose of this paper is to show that the variance effect found by Cooper et al. (their Fig. 5) arises because the transformation from the raw data scale (14 bits) to the display scale (12 bits), for the digital mammography system they investigated, is not logarithmic for raw data values less than about 300 (display data values greater than about 3300). At low raw data values the transformation is linear and prevents over-ranging of the display data scale. Parametric models for the two transformations will be presented. Results of pixel variance measurements made on raw data images will be presented. The experimental data are in good agreement with those of Cooper et al. It will be shown that the slope of 0.6 found by Cooper et al. for the log-log plot at low exposure is not due to transfer function nonlinearity, it occurs because of an additive variance term-possibly due to electronic noise. It will also be shown, using population statistics from clinical images, that raw data values below 300 are rare in tissue areas. Those tissue areas with very low raw data values are within about a millimeter of the chest wall or in very dense muscle at comers of images.     

Electron microscopic analysis of two-dimensional crystals of the Ca2+-transport ATPase — a freeze-fracture study

Summary Two distinct forms of Ca²⁺-ATPase crystals have been analysed in sarcoplasmic reticulum (SR) membranes. The E₁-type crystals, induced by Ca²⁺ or lanthanide ions, consist of single chains of ATPase monomers, and the E₂-type crystals, induced by vanadate ions, consist of dimer chains. Using improved freeze-fracture techniques we have obtained high-resolution images of complementary surface replicas of SR membranes containing these crystal forms. In E₁ crystals, the concave fracture (P) faces display obliquely oriented rows of intramembrane particles (IMPs) spaced at - 6–7 nm along both crystal axes, while the convex fracture (E) faces show corresponding rows of pits. In E₂ crystals, regular arrays of oblique parallel ridges with spacing of - 10.5–11 nm appear on the P-faces and complementary grooves or furrows on the E-faces. In many instances the ridges break up into elongated particles repeating every 5.5 nm. When the direction of the shadow is almost parallel to the axis of the ridges, these 9.5 nm particles can be resolved into two domains, which represent intramembranous contacts between the two monomers of the two adjacent dimer chains. Complementary grooves on the E-faces can also be resolved into rows of pits complementary to the particles of the ridges on the P-faces. In the control SR membranes, randomly dispersed IMPs and corresponding pits are observed on the P- and E-faces, respectively. The data suggest that transport of Ca²⁺ involves significant structural changes of the enzyme molecule, reflected in the ATPase-ATPase interactions both on the cytoplasmic surface and in the lipid bilayer.     

Growth factor-induced phosphorylation of c-ras p21 in normal hemopoietic progenitor cells

Normal murine hemopoietic progenitor cells (colony-forming cells, CFC), representing 0.2% of the bone marrow cell population, were purified to homogeneity by fluorescence-activated cell sorting. CFC require the presence of the murine hemopoietic regulator, granulocyte-macrophage-colony stimulating factor (GM-CSF) for survival, proliferation, and differentiation along the myeloid pathway. An analysis of protein phosphorylation in GM-CSF-stimulated CFC over a 20-hr period demonstrated three phosphoproteins of approximate MW 21 kd and pI 6.2, 5.7 and 5.2 p21-6.2 persisted for 14 hr, while p21-5.7 and p21-5.2 were only detected during the first 5 hr of the analysis. The phosphate turnover time in all three p21 proteins was less than 3 hr and p21-5.2 contains an alkaline-resistant phosphorylation site. Low levels of p21-6.2 were also detected in unstimulated CFC. The observation of these phosphoproteins led us to investigate c-ras p21 in CFC. Immune precipitation with the anti-Ha/Ki-ras p21 monoclonal antibody (Y13-259) showed that expression of c-ras p21 in CFC was independent of GM-CSF stimulation, but that phosphorylated c-ras p21 was present only after GM-CSF stimulation. CFC contained one-tenth of the amount of phosphorylated c-ras p21 per cell compared with v-Ha-ras-transformed fibroblasts. It is possible that the phosphorylation of c-ras p21 in CFC has a significant role in the growth factor-directed molecular cascade responsible for normal hemopoietic development.     

Human granulocyte-macrophage colony-stimulating factor (hGM-CSF): identification of a binding site for a neutralizing antibody.

One approach to the localization of functionally active regions of human granulocyte-macrophage colony-stimulating factor (hGM-CSF) is to map the epitopes recognized by neutralizing anti-hGM-CSF monoclonal antibodies. We have defined the epitope recognized by one neutralizing antibody (LMM102) using proteolytic fragments obtained by enzymic digestion of bacterially synthesized hGM-CSF. RP-HPLC fractionation of a tryptic digest resulted in the identification of an immunoreactive "tryptic core" peptide containing 66 amino acids (52% of the protein). Further digestion of this "tryptic core" with S. aureus V8 protease produced a unique immunoreactive hGM-CSF product comprising two peptides, residues 86-93 and 112-127, linked by a disulfide bond between residues 88 and 121. The individual peptides, generated by reduction with dithiothreitol, were not recognized by the antibody. An analog of this peptide has been synthesized chemically and shown to have similar immunoreactivity to the epitope obtained by enzymic digestion. A series of modified peptides has also been synthesized to identify further the region required for antibody recognition.