Induction of congenital anomalies in offspring of female mice exposed to varying doses of X-rays

Female mice were exposed to varying absorbed doses (108–504 rad) of X-rays and mated at different intervals after irradiation (1–7, 8–14, 15–21 and 22–28 days). Uterine contents were examined at late pregnancy in order to detect early fetal deaths (dominant lethality) and malformations in the live fetuses.Two trends were apparent from data on abnormal fetuses. At each weekly interval, the incidence of abnormalities tended to rise with increase in dose, and, at any given dose, the incidence tended to increase with time after irradiation. Dwarfism and exencephaly were the two most common malformations found.The changes in incidence of dominant lethality and of abnormal fetuses with time and with dose follow each other closely, the highest incidence for both being reached in week 3 (59±4.7% for dominant lethals and 12.5±3.1% for abnormal fetuses, after 504 rad) indicating increased radiosensitivity of less mature oocytes. These results parallel those obtained from known genetic effects reported by other workers and suggest that testing for incidence of congenital malformations among offspring of treated animals may prove a useful means of assessing genetic hazards of radiation of chemicals.     

Quercetin modulates keratoconus metabolism in vitro

Corneal scarring is the result of a disease, infection or injury. The resulting scars cause significant loss of vision or even blindness. To‐date, the most successful treatment is corneal transplantation, but it does not come without side effects. One of the corneal dystrophies that are correlated with corneal scarring is keratoconus (KC). The onset of the disease is still unknown; however, altered cellular metabolism has been linked to promoting the fibrotic phenotype and therefore scarring. We have previously shown that human keratoconus cells (HKCs) have altered metabolic activity when compared to normal human corneal fibroblasts (HCFs). In our current study, we present evidence that quercetin, a natural flavonoid, is a strong candidate for regulating metabolic activity of both HCFs and HKCs in vitro and therefore a potential therapeutic to target the altered cellular metabolism characteristic of HKCs. Targeted mass spectrometry‐based metabolomics was performed on HCFs and HKCs with and without quercetin treatment in order to identify variations in metabolite flux. Overall, our study reveals a novel therapeutic target OF Quercetin on corneal stromal cell metabolism in both healthy and diseased states. Clearly, further studies are necessary in order to dissect the mechanism of action of quercetin. Copyright © 2015 John Wiley & Sons, Ltd.     

Hepatocyte growth factor/scatter factor and its interaction with heparan sulphate and dermatan sulphate

Hepatocyte growth factor (HGF)/scatter factor (SF) is a unique growth factor, in that it binds both heparan sulphate (HS) and dermatan sulphate (DS). The sequences in HS and DS that specifically interact with and modulate HGF/SF activity have not yet been fully identified. Ascidian DS, which uniquely possesses O-sulphation at C-6 (and not C-4) of its N -acetylgalactosamine unit, was analysed for HGF/SF-binding activity in the biosensor. The kinetic analysis revealed a strong, biologically relevant interaction with an equilibrium dissociation constant ( K (d)) of approx. 1 nM. An Erk activation assay also demonstrated stimulation of the MAP kinase pathway downstream of the Met receptor following addition of both HGF/SF and ascidian DS to the glycosaminoglycan-deficient CHO-745 mutant cell line. Furthermore, the activation of Met and the MAP kinase pathway by HGF/SF and ascidian DS leads to a cellular response in the form of migration.     

Specific chemical modifications of link protein and their effect on binding to hyaluronate and cartilage proteoglycan

Specific chemical modifications of amino acid residues were performed on purified, native link protein from bovine articular cartilage. The effects of these on link protein's interactions with hyaluronate and bovine articular cartilage proteoglycan were assayed by gel chromatography. Interaction with hyaluronate was significantly perturbed by modification of lysine, arginine, tyrosine and aspartic/glutamic acid residues, but not histidine and tryptophan residues. No free, accessible sulphydryl group was found on native link protein. The requirement for unmodified lysine and arginine residues resembles that of the hyaluronate-binding site of pig laryngeal cartilage proteoglycan (Hardingham, T.E., Ewins, R.J.F. and Muir, H. (1976) Biochem. J. 157, 127-143). In contrast, proteoglycan binding was only significantly perturbed by the loss of arginine residues. This resistance may reflect hydrophobicity of the binding site or masking of the site from chemical modification by link protein self-association. Amidation of carboxyl groups, which destroyed hyaluronate binding but left proteoglycan binding intact, provides a means of generating a monofunctional link protein molecule of potential use in proteoglycan aggregation studies.     

The impact of different fixation procedures on staining of macromolecules in a microtiter system

Summary The effects of formaldehyde, glutaraldehyde, methanol, Clarke's fixative and microwave irradiation on the quantitative staining of proteins (Naphthol Yellow S) and nucleic acids (Ethyl Green—Pyronin) in a cell culture system have been investigated. Overall, glutaraldehyde rapidly yielded the highest and most consistent levels of staining when compared to all other chemical fixatives. Although microwave irradiation was found to be uneven, 4 min exposure to 700W was found to give higher levels of protein staining than those achieveable with glutaraldehyde. Time-dependent processes were observed with all procedures. In addition, dissociations in the trends of protein and nucleic acid staining were observed. It is suggested that these results domonstrate fixation events that have not previously been resolved from the effects of reagent penetration into tissue blocks.