Quantification and Topographical Distribution of Terminal and Linked Fucose Residues in Human Spermatozoa by Using Field Emission Scanning Electron Microscopy (FE-SEM)

The modification of sperm glycocalyx is an essential process during sperm capacitation. The presence and redistribution of terminal and linked fucose have been described during in vitro capacitation in humans. However, the influence of the capacitation time on the quantification and localization of terminal and linked fucose is still unknown. In this study, the quantitative and qualitative changes in fucosyl residues during different in vitro capacitation times (1 and 4 h), are simultaneously characterized by using Aleuria aurantia (AAA) lectin–gold labelling and high-resolution field emission scanning electron microscopy (FE-SEM) in human sperm. A significant decrease was found in the number of terminal fucose registered in the whole sperm head during the in vitro capacitation. Nevertheless, the quantification of fucose residues after 1 h of in vitro capacitation was very similar to those found after 4 h. Therefore, the changes observed in terminal and linked fucose during capacitation were not time-dependent. Furthermore, the comprehensive analysis of the topographic distribution showed the preferential fucosyl location in the acrosomal region and the presence of distinct clusters distributed over the head in all the studied conditions. Overall, these findings corroborate the validity of FE-SEM combined with gold labelling to register changes in surface molecules during in vitro sperm capacitation.

Gold labelling of Green Fluorescent Protein (GFP)-tag inside cells using recombinant nanobodies conjugated to 2.4 nm thiolate-coated gold nanoparticles

The advances in the microscopy technology have prompted efforts to improve the reagents required to recognize specific molecules within the intracellular environment. For high-resolution electron microscopy, conjugation of selective binders...

A lateral flow immunochromatographic strip test for rapid detection of hexoestrol in fish samples

A lateral flow immunochromatographic strip test was developed for rapid and sensitive on-site detection of hexoestrol (HES) residues in fish samples with colloidal gold labelling of the anti-HES monoclonal antibody. The strip is composed of a sample pad, a conjugate reagent pad, an absorbent pad and a test membrane containing a control line and a test line. The sensitivity (half inhibitory concentration, IC 50 ) of the strip in the detection of fish extract samples was confirmed to be 1.86 µg kg −1 , and the limit of detection value was 0.62 µg kg −1 . For intra-assay and inter-assay reproducibility, recoveries of HES-spiked samples ranged from 86.3% to 92.3% and 85.8% to 93.4%, coefficients of variation were 2.91–4.64% and 4.24–5.17%, respectively. High-performance liquid chromatography was employed to confirm the performance of the strip. The strip test takes less than 10 min, and thus provides a repaid method for on-site detection of HES residues.

Premyelinated Central Axons Express Neurotoxic NMDA Receptors: Relevance to Early Developing White-Matter Injury

Ischemic-type injury to developing white matter is associated with the significant clinical condition cerebral palsy and with the cognitive deficits associated with premature birth. Premyelinated axons are the major cellular component of fetal white matter and loss of axon function underlies the disability, but the cellular mechanisms producing ischemic injury to premyelinated axons have not previously been described. Injury was found to require longer periods of modelled ischemia than at latter developmental points. Ischemia produced initial hyperexcitability in axons followed by loss of function after Na+ and Ca2+ influx. N-methyl-D-aspartate- (NMDA) type glutamate receptor (GluR) agonists potentiated axon injury while antagonists were protective. The NMDA GluR obligatory Nr1 subunit colocalized with markers of small premyelinated axons and expression was found at focal regions of axon injury. Ischemic injury of glial cells present in early developing white matter was NMDA GluR independent. Axons in human postconception week 18 to 23 white matter had a uniform prediameter expansion phenotype and postembedded immuno-gold labelling showed Nr1 subunit expression on the membrane of these axons, demonstrating a shared key neuropathologic feature with the rodent model. Premyelinated central axons therefore express high levels of functional NMDA GluRs that confer sensitivity to ischemic injury.

Molecular directionality in crystalline β-chitin: hydrolysis by chitinases A and B from Serratia marcescens 2170

β-Chitin microfibrils were treated with ChiA and ChiB (chitinases A and B respectively) from Serratia marcescens 2170. The β-chitin microfibrils were shortened, and the tips appeared narrowed and sharpened at both ends, after either consecutive or simultaneous degradation by ChiA and ChiB. Increased production of reducing sugars by simultaneous degradation (by ChiA and ChiB) of β-chitin, but not of glycol chitin, suggests synergistic interactions between the two enzymes. A combined analysis using the tilt microdiffraction method to determine the crystallographic axes, together with the biotin–streptavidin–gold-labelling method specific to the reducing ends, was used to investigate the polarity of the degraded β-chitin microcrystals. The digestion of the β-chitin fibrils by ChiA occurred from the reducing end to the nonreducing end, whereas digestion by ChiB occurred from the non-reducing end to the reducing end. The results are in agreement with the previously determined three-dimensional structures of these enzymes.