The Release of a Soluble Glycosylated Protein from Glycogen by Recombinant Lysosomal α-Glucosidase (rhGAA) In Vitro and Its Presence in Serum In Vivo

In studies on the degradation of glycogen by rhGAA, a glycosylated protein core material was found which consists of about 5–6% of the total starting glycogen. There was an additional 25% of the glycogen unaccounted for based on glucose released. After incubation of glycogen with rhGAA until no more glucose was released, no other carbohydrate was detected on HPAEC-PAD. Several oligosaccharides are then detectable if the medium is first boiled in 0.1 N HCl or incubated with trypsin. It is present in serum either in an HCl extract or in a trypsin digest. The characteristics of the in vivo serum material are identical to the material in the in vitro incubation medium. One oligosaccharide cannot be further degraded by rhGAA, from the incubation medium as well as from serum co-elute on HPAEC-PAD. Several masked oligosaccharides in serum contain m-inositol, e-inositol, and sorbitol as the major carbohydrates. The presence of this glycosylated protein in serum is a fraction of glycogen that is degraded outside the lysosome and the cell. The glycosylated protein in the serum is not present in the serum of Pompe mice not on ERT, but it is present in the serum of Pompe disease patients who are on ERT, so it is a biomarker of GAA degradation of lysosomal glycogen.

The Effects of In Vitro Incubation of Asthenoteratozoospermic Semen after Density Gradient Centrifugation at Room Temperature and 37C on Sperm Parameters, Chromatin Quality and DNA Fragmentation in a Short Time Period

Background: Sperm quality is an important factor in assisted reproductive technology (ART) that affects the success rate of infertile couples treatment. In vitro incubation of sperm can influence its parameters and DNA integrity. The present study focused on the effect of different incubation temperatures sperm parameters on asthenoteratozoospermia semen prepared with density gradient centrifugation at different times. Methods: Twenty-seven samples were collected and prepared. Then, the suspension was divided into two parts. One part was incubated at room temperature (RT), and another was incubated at 37°C. Immediately and after 2 hr (2H) and 4 hr (4H), sper-matozoa were evaluated regarding motility, viability, morphology, sperm protamine deficiency, chromatin and DNA fragmentation. Statistical analysis was performed using paired t-test and repeated measures. The p<0.05 was considered statistically significant. Results: Our results showed that following 2 and 4 hr of incubation at RT, sperm progressive motility and viability decreased significantly. Sperm DNA fragmentation increased significantly following 2 and 4 hr of incubation at RT and 37°C. The Trend analysis confirmed that there were no significant differences between sperm parameters and DNA fragmentation after different times at RT and 37°C. Conclusion: Incubation of sperm at RT in comparison to 37°C didn’t preserve sperm parameters and DNA efficiently. Therefore, IVF, ICSI and IUI procedure should be performed in the soonest possible time after sperm preparation.

In vitro and in situ techniques yield different estimates of ruminal disappearance of barley

Objectives were to compare in vitro and in situ disappearance of dry matter (DM), neutral detergent fiber (NDF), and starch of traditional (unprocessed and rolled) and hulless (unprocessed) barley. Experiment 1: three barley sources were compared using in vitro techniques. The sources were: 1) traditional barley that was not processed, 2) traditional barley processed through a roller mill, and 3) hulless barley that was not processed. For in vitro incubation, each barley source was ground through a 1-mm screen. Ground barley sources were weighed into bags (25 micron porosity) and incubated in ruminal fluid from two steers fed 80% rolled corn for 3, 6, 12, 24, 48, or 72 h. Intact bags were assayed for NDF; remaining bags were opened and the residual was removed and analyzed to determine disappearance of DM and starch. Experiment 2: the barley sources used in Exp. 1 were compared using in situ techniques. For in situ analysis, each barley source was ground in a Wiley mill with no screen to mimic mastication. Artificially masticated samples were weighed into Dacron bags (50 ± 10 micron porosity) and incubated in eight ruminally fistulated steers (n = 8) for 3, 6, 12, 24, 48, and 72 h. Residual contents were analyzed to determine in situ disappearance of DM, NDF, and starch. Data were analyzed using the MIXED procedures of SAS (9.4 SAS Institute, Cary, NC) with repeated measures. DM disappearance was greatest (P &lt; 0.05) for hulless barley in vitro and for rolled barley in situ, regardless of time postincubation. For both trials, NDF disappearance was greatest (P &lt; 0.05) for hulless barley, regardless of time postincubation. Starch disappearance at all time points was greatest (P &lt; 0.05) for rolled barley in situ. Starch disappearance was greater (P &lt; 0.05) for hulless barley at 6 h of in vitro incubation compared to rolled and unprocessed barley, whereas starch disappearance in vitro was comparable (P = 0.60) between barley sources. When the grains were compared in vitro, minor differences were noted, presumably because barley sources were finely ground prior to incubation. Compared to in vitro estimates, in situ techniques had greater variation in ruminal degradation estimates. Differences observed between in situ and in vitro techniques are driven largely by differences between the procedures. Although laboratory methods are widely used to estimate ruminal degradation, these techniques did not provide comparable estimates of ruminal degradation of barley.